XN-Series Flagging Interpretation Guide
Version 2
Date:
Subject:
Issued by:
Version:
14 April 2022
Interpretation guide for XN-Series IP messages
Product Management Haematology & Integrated Laboratory Solutions
2.0
Contents
1
2
Objectives..........................................................................................................................................................5
XN Analytical Methods .....................................................................................................................................5
Hydrodynamically focussed impedance measurement in RBC/PLT channel .................................................5
Cyanide-free SLS haemoglobin measurement ...............................................................................................6
Fluorescence flow cytometry in WNR, WDF, RET, PLT-F and WPC channels ..............................................7
3 XN-Series flagging and messages ..................................................................................................................8
General definitions ..........................................................................................................................................8
IP messages ....................................................................................................................................................9
Positive / Negative judgement .........................................................................................................................9
Q-Flags .........................................................................................................................................................10
4 WBC flagging ..................................................................................................................................................12
Scattergram patterns .....................................................................................................................................12
WBC abnormal flags......................................................................................................................................13
‘WBC Abn Scattergram’ in WNR channel .....................................................................................................13
‘WBC Abn Scattergram’ in WDF channel ......................................................................................................16
‘WBC Abn Scattergram’ in BF mode .............................................................................................................19
‘NRBC Present’ .............................................................................................................................................19
‘IG Present’ ....................................................................................................................................................20
WBC suspect flags ........................................................................................................................................21
‘Left Shift?’ .....................................................................................................................................................21
A brief overview: How does the XN manage the appearance of pathologic cells in the WDF ? ...................22
‘Blasts/Abn Lympho?’ ....................................................................................................................................24
‘Blasts?’ .........................................................................................................................................................25
‘Abn Lympho?’ ...............................................................................................................................................26
Blasts AND Abnormal Lympho? ....................................................................................................................27
‘Atypical Lympho?’.........................................................................................................................................28
5 RBC flagging ...................................................................................................................................................29
RBC histogram patterns ................................................................................................................................29
RBC abnormal flags ......................................................................................................................................29
‘RBC Abn Distribution’ ...................................................................................................................................29
‘Dimorphic Population’ ...................................................................................................................................30
RBC suspect flags .........................................................................................................................................31
‘Turbidity/HGB Interf? ....................................................................................................................................31
RBC Agglutination?’ ......................................................................................................................................31
‘Iron Deficiency?’ ...........................................................................................................................................33
‘HGB Defect?’ ................................................................................................................................................33
‘iRBC?’ ..........................................................................................................................................................34
6 RET flagging....................................................................................................................................................35
RET channel scattergrams ............................................................................................................................35
RET abnormal flag.........................................................................................................................................35
‘RET Abn Scattergram’ ..................................................................................................................................35
RET suspect flags .........................................................................................................................................37
‘Fragments?’ ..................................................................................................................................................37
7 PLT flagging ....................................................................................................................................................38
PLT histogram from RBC/PLT channel .........................................................................................................38
PLT-F channel scattergram ...........................................................................................................................38
PLT abnormal flags .......................................................................................................................................38
‘PLT Abn Distribution’ ....................................................................................................................................38
‘PLT Abn Scattergram’ ..................................................................................................................................39
PLT suspect flags ..........................................................................................................................................40
‘PLT Clumps?’ ...............................................................................................................................................40
‘Giant Platelet?’ .............................................................................................................................................42
8 Action messages ............................................................................................................................................43
9 Possible sample interferences ......................................................................................................................46
10 Supporting literature ......................................................................................................................................47
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2
© 2022, Sysmex Europe SE. All rights reserved.
This document contains information that could support the user to interpret IP (Interpretive Program) messages
generated by the XN-Series analysers. IP Messages are only intended for use in the clinical laboratory and are
not for patient diagnosis. It is not intended to substitute the information available in the Instructions for Use (IFU)
and Administrator’s Guide (ADM) but as supplementary material. In case of any editorial errors or omissions
contained herein, the reference document is always the IFU and/or the ADM.
Sysmex is a registered trademark of Sysmex Corporation.
This guide was created by Sysmex Europe SE. Kindly direct your questions and comments regarding the
content to us via your local Sysmex representative.
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3
Revision history
No
Page
number
Revised content in version 2
1
Naming of measurement channels
5, 6 and 7
2
Modified text regarding logic to assign Q-flag value
11
3
Note on ‘NRBC present’ IP message and its relation to ‘WBC Abn Scattergram’ flag
13
4
Added content regarding changes applied with SW 22.15 under ‘Details’
14
5
Added new condition under triggers for ‘WBC Abn Scattegram’ from WDF channel
16
6
Added examples / details of ‘Low SFL rule’ in WDF scattergram
18
7
Deleted contents under ‘WBC Abn Scattegram’ in BF mode that were related to SW 21.12, as the
SW is no longer new
19
8
Updated definition of AFLAS, modified WDF scattergram depicting the AFLAS
22
9
Adapted image depicting the two-step approach to exclude malignant samples with WDF/WPC
23
10
Added an example for the IP messages ‘Blasts?’ and ‘Abn Lympho?’ after a WPC measurement
27
11
Corrected ‘’The RBC histogram plots event size…’ to ‘’The RBC histogram plots event volume…’
29
12
Adapted ‘Suggested action’ for the IP message ‘iRBC?’
35
13
Inserted condition and examples related to ‘if there is no clear separation between the RBC/RET
and PLT-O clusters’ under ‘RET Abn Scattergram’
36
14
Corrected / updated scattergram examples for ‘RET Abn Scattergram’
36
15
Added explanation for internal parameters for the IP message ‘Fragments?’
37
16
Added WNR (FSCW-FSC) scattergram
40
17
Updated text under ‘Suggested actions’
41
18
Updated text under ‘Giant Platelet?’ IP message – ‘The ‘Giant Platelet?’ flag was implemented
with SW 21.12 onwards and is part of the ‘PLT Clumps?’ flagging strategy
42
19
Added definitions for the different types of ‘Action messages’
43
20
Updated ‘Action messages’ table
45
21
Updated ‘Possible sample interferences’ in accordance with the latest XN-Series IFU
47
22
Various images and tables throughout the document were renewed.
-
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4
1
Objectives
The flagging guide is designed to serve many objectives. These include providing you with:
◼ brief functional descriptions of the measurement principles of the different channels
✓
Hydrodynamically focussed impedance measurement
✓
Cyanide-free SLS haemoglobin measurement method
✓
Fluorescence flow cytometry
◼ an overview of the different types of Interpretive Program (IP) messages
✓
An explanation of the criteria used for the XN-Series IP messages that are not operator programmable.
✓
Suggested actions to be taken when samples generate IP messages.
✓
Suggested actions to resolve sample-related problems.
The XN-Series software is designed to aid in separating specimens into POSITIVE and NEGATIVE categories
according to pre-set criteria. The system bases its judgments on comprehensive surveys of numerical data,
particle size distributions, scattergrams and provides easy-to-understand flags/messages that indicate the
instrument's findings. These flags and messages are referred to as IP messages.
A specimen is judged NEGATIVE when there are no predefined abnormalities present in the sample. The results
are generally reported without review.
The XN-Series analysers will generate a POSITIVE judgement when an IP message is present. An established
review process by lab personnel should be initiated.
POSITIVE or ERROR judgments indicate the possibility of a sample abnormality. These results should be
reviewed carefully and may require further examination in accordance with the laboratory standard operating
protocol (SOP).
Some of the action steps suggested in this guide may coincide with procedures previously established and
implemented in a lab. These action steps are merely suggested guidelines and should be used in conjunction
with the laboratory’s SOP.
2
XN Analytical Methods
Hydrodynamically focussed impedance measurement in RBC/PLT channel
◼ This method is used in every CBC analysis.
◼ Volumetric measurement of red blood cells and platelets using absolute counting by DC detection method
with hydrodynamic focusing (HDF).
◼ A diluted sample is ejected from the nozzle tip and the blood cells enclosed in sheath fluid pass through a
defined path at the centre of the aperture, as depicted below.
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5
◼ As each blood cell passes through the centre of the aperture, an electric resistance proportional to the
volume of that blood cell is created.
Electric resistance
Cell volume
◼ This information is plotted as a histogram, and deviations from the expected results trigger IP message(s).
PLT
RBC
PLT
RBC
Cyanide-free SLS haemoglobin measurement
◼ This method is also used in every CBC analysis.
◼ The SLS haemoglobin method is based on cyanide-free reagents using sodium lauryl sulphate (SLS).
◼ The haemoglobin measurement follows the reaction steps shown in the figure below.
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✓
The surfactant lyses the RBC to release the haemoglobin molecules.
✓
Hydrophobic groups of SLS act upon the globin molecules, changing their conformation.
✓
The iron of the molecules is oxidised (Fe2+ to Fe3+).
✓
Hydrophilic groups of SLS bind with Fe3+ forming a stable colourimetric complex.
◼ The haemoglobin concentration is determined from the absorbance measured by a photometric method at a
wavelength of 555 nm as shown below.
Photo sensor
Flow cell
Lysed blood sample
LED with filter
Fluorescence flow cytometry in WNR, WDF, RET, PLT-F, and WPC channels
◼ The following icons indicate that fluorescence flow cytometry is the key technology used in WNR, WDF, RET,
PLT-F, and WPC channels.
◼ Fluorescence flow cytometry (FFC) is used to analyse cells' physiological and structural properties while they
are flowing through a very narrow flow cell.
◼ First, a blood sample is aspirated and proportioned, then diluted to a pre-set ratio and labelled with a
proprietary fluorescent marker that binds specifically to nucleic acids.
◼ Next, the sample is transported into the flow cell. The sample is illuminated by a semiconductor laser beam,
which can separate the cells using three different signals, each of which provides unique information:
✓
Forward-scattered light (FSC) - cell volume.
✓
Side-scattered light (SSC) - cell content, such as the nucleus and granules.
✓
Side-fluorescence light (SFL) - the amount of nucleic acids and cell organelles.
◼ These three signals, highlighted in the image below, are used to count and differentiate WBC, nucleated RBC
(NRBC), reticulocytes, and PLT, and to detect immature and abnormal cells based on unique algorithms.
Side scattered light
receiver
Side fluorescent
light receiver
Forward scattered
light receiver
Dichroic
mirror
Flow cell
Semiconductor
laser
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3
XN-Series flagging and messages
General definitions
4
3
5
1
1
6
2
No
Flagging and messages
Marks
Only one mark can be appended
per data value. If multiple
abnormalities apply to a parameter,
whichever abnormality has the
highest priority is notated. Priorities
are assigned to the marks in the
order they appear in the table.
1
Notations
Meaning
[*]
Low reliability
[@]
Out of range
[!]
Exceeds upper or
lower panic limit /
exceeds upper
acceptable
background check
limit
[+]
Exceeds upper limit
[-]
Exceeds lower limit
Only the priority levels of [*] and [@]
can be changed in the service
settings.
Description
This indicates that the reliability of the data is low.
This indicates that the data is outside the linearity
limits.
This indicates that the value is higher than or less
than the clinical panic value. Also indicates that
the value is higher than the allowed value for a
background check.
Depends on critical value settings.
This indicates that the value is higher than the
reference interval.
Depends on reference value settings.
This indicates that the value is less than the
reference interval.
Depends on reference value settings.
Masks
2
[----]
Analysis impossible
Indicates that an analysis error or a parsing error
has occurred, and the value cannot be displayed.
[++++]
Out of range
This indicates that the data cannot be displayed
because the value exceeds the display limit.
[]
No order
This indicates that the analysis order does not
exist.
IP messages
3
-
-
Provides a notification of the possibility of a
specific sample abnormality based on
examination of the analysis data.
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8
Q-Flags
4
For more information, please refer
to page 10
-
-
-
-
Action messages
5
6
Indicates the Positive/Negative levels for suspect
IP messages.
Displays an action message if one exists.
For more information, please refer
to page 43
Error/Rule messages(Rerun/reflex
rules may be deactivated if the XN
configuration is connected to the
Extended IPU)
-
-
Displays the error message and/or rule comment
if one exists. The rule comments are sorted by
priority, with the highest priority on top, and then
by rule number in ascending order.
IP messages
Message types
◼ There are two types of IP messages, abnormal messages and suspect messages, that may be displayed for
WBC, RBC/RET, and PLT.
✓
Abnormal message: indicates that the sample is clearly abnormal. With some exceptions, the user can predefined the criteria for the ‘abnormal message’ judgment.
✓
Suspect message: indicates a possibility that the sample is abnormal.
Positive / Negative judgement
◼ [Positive] Indicates that an analysis value or cell morphology exceeds the predefined criteria for the IP
message (abnormal sample). It is displayed on a red background.
◼ A Positive judgment can be classified into three types of abnormalities:
✓
[Diff.]: indicates an abnormal blood cell differentiation value.
✓
[Morph.]: indicates abnormal cell morphology.
✓
[Count]: indicates an abnormal blood cell count.
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9
◼ [Negative] Indicates that there was no analysis error or abnormality detected by the analyser. Hence, there is
no IP message (normal sample). It is displayed on a green background.
Q-Flags
The [Q-Flag] screen displays the Positive/Negative levels for suspect IP messages as a bar graph. The shown
information corresponds to the sample you selected in the [Sample Explorer] screen.
[Discrete] as the related
parameters were not analysed
Negative Q-Flag when
the value falls below
the threshold.
Positive Q-Flag when the
value exceeds the threshold
Pre-conditions for judgement were
not met, so the Q-Flag is blank
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◼ In the bar graph, the negative results of the sample are displayed in green, and the positive results are shown
in red.
◼ The values in the bar graph range from 0 to 300, in increments of 10. Values over 100 (threshold) are
determined as positive.
◼ In addition, the following may appear in the judgment value position (when the bar graph is empty):
✓
[Discrete]: displayed in grey text. If the parameter used for the judgment was not analysed.
✓
[Error]: if the judgment was not possible.
✓
[
]: Blank appears if a prerequisite for the judgment was not met. Also, if the suspect judgment was not
performed due to blank data, etc.
◼ The threshold for the different Q-Flags should normally never be changed (except if indicated by Sysmex
Europe SE).
◼ The value for almost every Q-Flag is semi-quantitative. It mainly reflects the degree of the specific
abnormality for more efficient screening. RBC-related flags, including ‘Turbidity/HGB Interf?’, ‘Iron
Deficiency?’ and ‘HGB Defect?’ are triggered by a single rule considering different RBC parameters.
◼ WBC-related suspect flags such as ‘Blasts/Abn Lympho? are assigned the highest score based on several
disparate rules, some of which consider cell counts while others consider ratio or cell cluster positions. On the
chronic lymphocytic leukaemia (CLL) example below, the XN analyser judged for ‘Blasts/Abn Lympho?’ within
the lymphocyte branch. In this case, the Q-flag value was calculated based on the lymphocyte count
(LYMPH#). The high ‘Blasts/Abn Lympho?’ Q-flag value of 260 is based on the elevated lymphocyte count.
WDF scattergram (SSC-SFL)
LYMPH# 7,83 x10³/µL
LYMPH% 54.8 %
Q-Flag value for ‘Blasts/Abn
Lympho?’ flag is 260
◼ A ‘Blasts/Abn Lympho?’ Q-Flag of 200 does not mean double the number of blasts compared to a Q-Flag of
100 but reflects a more abnormal WDF scattergram.
◼ The ’Blasts?’ and ‘Abn Lympho?’ Q-Flag value from the WPC channel is always the same as the
corresponding Q-Flag value for ‘Blasts/Abn Lympho?’ from the WDF channel.
◼ An exception amongst the WBC-related suspect flags is the ‘Atypical Lympho?’ flag, which correlates to the
RE-LYMP% with XN-Series SW 21.12 onwards.
◼ More details on the judgement criteria underlying suspect IP messages (and the associated Q-Flags) are
individually explained in the next chapters.
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11
4
WBC flagging
Scattergram patterns
Profiles
Normal scattergram patterns
Scattergrams highlighting the typical regions with abnormal
cells/ interferences
* Please note that the ‘Blast or LYMPH’ area in WPC (SSC-FSC) includes abnormal lymphocytes.
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12
WBC abnormal flags
‘WBC Abn Scattergram’ in WNR channel
SEU (Sysmex Europe SE) recommends enabling ‘Diff masking’.
(For more information, please contact your local Sysmex representative).
Details:
The ‘WBC Abn Scattergram’ IP message in the WB mode can be triggered
by abnormalities detected in the WNR and/or WDF scattergram.
In the WNR channel, some examples of abnormalities judged include:
◼ The different clusters in the WNR scattergram cannot be differentiated.
For example, separation of NRBC and WBC clusters is not possible.
◼ WBC aggregates have been detected in the WNR channel (FSCW-FSC
scattergram, which can be viewed on the ‘User’ or ‘Lab. Only’ screens).
◼ The appearance of abnormal particles extending from the low FSC area
in the WNR scattergram.
NOTE: If the IP message ‘NRBC present’ is switched OFF, but NRBC%
exceeds the threshold value, ‘WBC Abn Scattergram’ will be triggered
instead. Refer to page 19 for more details.
Suggested actions:
◼ Dashes [----] in place of the data:
✓
Check if alternative data is available (e.g. WBC-D instead of WBC-N,
if reliable).
✓
Perform a manual differential.
◼ Asterisk [*] next to the data:
WNR scattergram (SFL-FSC)
WNR scattergram (FSCW-FSC)
WNR scattergram (SFL-FSC)
✓
Scan the slide for abnormal cells.
✓
If no abnormalities are found, the data with asterisks [*] can be
reported.
✓
If abnormalities are found, perform a manual differential.
◼ Asterisk [*] next to the data and ‘greyed-out’ clusters in the scattergram:
✓
Overlapping NRBC
and WBC clusters
WBC aggregates in
the detection area
Perform a manual differential.
Interfering particles in
the low FSC area
When a sample judgment is positive for ‘WBC Abn Scattergram’, dashes
may appear in place of data that was not calculated, so-called masking
[----]. ‘Diff masking’ can be enabled/disabled in the ‘Service Setting’. If ‘Diff
masking’ is not enabled, the results may be available as unreliable with an
asterisk [*].
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13
Some detailed examples of ‘WBC Abn Scattergram’ in the WNR channel
Suggested actions:
Overlapping NRBC and WBC clusters
◼ Check for TNC-N data in the ‘Lab. Only’ screen.
WNR scattergram (SFL-FSC)
WNR scattergram (SFL-FSC)
◼ WBC count can be derived from the TNC-N count by subtracting the
NRBC count. NRBC count must be derived manually.
◼ Subtract the NRBC from the TNC according to your laboratory SOP.
◼ Perform a manual differential.
SW 00-18
SW 21.12
Details:
◼ WBC and NRBC populations cannot be separated sometimes due to
highly immature NRBC, which overlap with the WBC population.
◼ WBC results could be falsely high in such cases.
◼ WBC and NRBC#/% are masked [----] (if ‘DIFF masking’ is ON); other
WBC related parameters can also be marked/masked in XN-Series SW
versions prior to SW 22.15. With SW 22.15 and onwards, the NRBC
and relevant WBC-related parameters are masked [----] independent of
the ‘DIFF masking’ setting.
◼ Such cases are detected based on the unusual shape and position of
the WBC population, due to the inclusion of the NRBC.
◼ Commonly seen in case of neonate blood, haemolytic anaemia,
extreme recovery from iron deficiency, Thalassaemia (and other RBC
diseases) and leukaemia.
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14
WBC aggregates in the detection area
WNR scattergram (FSCW-FSC)
Interfering particles in the low FSC area
WNR scattergram (SFL-FSC)
Details:
Details:
WBC aggregates were detected in the WNR (FSCW-FSC) scattergram
(‘User’ or ‘Lab. Only’ screen).
Interference of particles extending from the low FSC area.
◼ Calculation of WBC-N is not possible due to WBC aggregates.
◼ When the WDF channel is measured, WBC-N switches to WBC-D,
indicated by WBC &D, and WBC-N and NRBC%/# are masked [----].
◼ In this case, abnormal particles are reclassified as ‘Ghost’, the lowreliability mark [*] is added to WBC-N and all WBC-related parameters
(including the WBC differential) and NRBC#/%.
Suggested actions:
Suggested actions:
◼ Scan the slide for NRBC.
◼ If NRBC is present, perform a manual differential. If WBC-D is available,
manually correct the WBC-D count according to your laboratory SOP.
◼ Check if alternative data is available (e.g. WBC-D instead of WBC-N, if
reliable).
◼ After ruling out any interference in the WDF scattergram, report
WBC-D.
◼ If no NRBC are detected, and interference is ruled out in the WDF
scattergram, report WBC-D and WBC differential.
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15
‘WBC Abn Scattergram’ in WDF channel
Suggested actions:
Details:
◼ Dashes [----] in place of data or asterisk [*] next to data
As mentioned, the ‘WBC Abn Scattergram’ IP message in the WB mode
can be triggered by abnormalities detected in the WNR and/or WDF
scattergram.
with ‘greyed-out’ clusters in the scattergram:
In the WDF channel, some common examples of abnormalities judged
include:
◼ Each cluster cannot be differentiated in the WDF scattergram as too
many particles are present on the border between two populations, e.g.
LYMPH: MONO border.
◼ 5-part DIFF calculation is not possible e.g. 100.0% < (LYMPH% +
MONO% + EO% + BASO% + NEUT%).
✓
Perform a manual differential.
◼ Asterisk [*] next to data:
✓
Scan the slide for abnormal cells.
✓
If no abnormalities are found, the data with asterisks [*] can be
reported.
✓
If abnormalities are found, perform a manual differential.
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
Cell clusters cannot be
differentiated
Abnormal NEUT cloud
◼ Abnormal slope, position and distribution of the neutrophil population on
the WDF scattergram.
◼ Low reliability of IG.
◼ Abnormally low SFL signal of the DIFF clusters in the WDF
scattergram.
When a sample judgment is positive for ‘WBC Abn Scattergram’, dashes
may appear in place of the data that was not calculated, termed masking
[----]. ‘Diff masking’ option can be enabled/disabled in the ‘Service Setting’.
If ‘Diff masking’ is not enabled, the affected parameters will be available as
unreliable with an asterisk [*].
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
SEU recommends enabling ‘Diff masking’.
(For more information, please contact your local Sysmex representative).
Unreliable IG classification
Abnormal low SFL of the
DIFF clusters
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16
Some detailed examples of ‘WBC Abn Scattergram’ in the WDF channel
Suggested actions:
Abnormal NEUT cloud
◼ Asterisk [*] next to data:
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
✓
Scan the slide for abnormal or immature cells.
✓
If no abnormalities are found, the data with asterisks [*] can be
reported.
✓
If abnormalities are found, perform a manual differential.
◼ Also, confirm that the WNR channel does not show any interferences
and the WBC-N value is reliable.
Cell clusters cannot be differentiated
Normal sample
Positive sample
Details:
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
◼ ‘WBC Abn Scattergram’ is triggered due to abnormalities of the
neutrophil population:
✓
Position on Y-axis.
✓
Distribution of the population.
✓
Shape (slope) of the population.
◼ Because of these anomalies, the analyser cannot identify with certainty
if the particles in that area are immature granulocytes, neutrophils, or
even unrelated interferences.
◼ Thus, the flag ‘WBC Abn Scattergram’ is triggered, and the IG and
NEUT parameters are considered unreliable [*].
◼ Examples of physiological triggers include an early bacterial infection
and sepsis/septic shock.
◼ Appears together with increased NEUT-RI (corresponds to NEUT-SFL).
SW 00-18
SW 21.12
Details:
◼ When the monocyte and lymphocyte populations cannot be correctly
differentiated, ‘WBC Abn Scattergram’ is triggered.
◼ The presence of a large ghost population could interfere with
lymphocyte and monocyte differentiation.
◼ The algorithm was revised with SW 21.12 to improve such cases.
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17
Suggested actions:
◼ Perform a microscopic differential.
Suggested actions:
◼ Also, confirm that the WNR channel does not show any interferences
and the WBC-N value is reliable.
Abnormally low SFL signal of the DIFF clusters
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
Low SFL signal e.g. Hb Mizuho
Low SFL signal e.g. met-Hb
◼ In such cases, WBC (WBC-N) is typically marked unreliable [*] due to
the action message ‘Difference between WNR and WDF. Check the
results. Verify the WNR scattergram to exclude interferences and then
use the WBC-N; perform a manual differential.
◼ Scan the slide for RBC abnormalities.
Details:
◼ Patient samples harbouring various unstable haemoglobin variants
typically show WDF scattergrams with a decrease in the fluorescence
signal. A similar pattern can be seen in samples with met-haemoglobin.
◼ Possible explanations for the cause of interference
✓
the high binding affinity of Hb variants to Fluorocell WDF
✓
the reduced permeability of the WBC cell membrane following
treatment with the surfactant reagent
✓
a combination of both.
◼ When the analyser detects such a low SFL signal, it can trigger the
‘WBC Abn Scattergram’.
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18
‘NRBC Present’
‘WBC Abn Scattergram’ in BF mode
‘WBC Abn Scattergram’ can also be triggered by abnormalities
judged in the analysis of BF samples.
WDF scattergram (SSC-SFL)
WNR scattergram (SFL-FSC)
WDF scattergram (FSCW-FSC)
Details:
Interfering liposome particles in different scattergram views
Details:
◼ The ‘NRBC Present’ IP message occurs when NRBC% is higher than
the predefined threshold.
Some of the abnormalities judged include:
◼ The threshold is user-defined and programmable; the default setting is
2%.
◼ WBC and ghost clusters cannot be separated.
◼ NRBC% are calculated as NRBC# / 100 WBC.
◼ Liposomal particles interfere with the WBC-BF scattergram (images
shown below).
◼ Physiologically, NRBC occurs in peripheral blood only in neonates and
premature babies.
◼ Presence of cells in the high fluorescence area (HF-BF). This is an
adjustable setting on the analyser. (For more information, please
contact your local Sysmex representative).
◼ In adults and older children, the occurrence of NRBC in circulating
blood always indicates serious disease. NRBC are frequently seen in
the case of haemolytic anaemias, thalassaemias, systemic
haematological diseases such as MDS or leukaemias, and severe
bleeding, but may also appear in a generally critical state of health, e.g.
trauma patients from intensive care units.
◼ If ‘WBC Abn Scattergram’ was triggered by the ghost or other
interference of WBC in body fluid analysis, TC-BF, WBC-BF, MN#/%
and PMN#/% will be marked unreliable [*].
Suggested actions:
✓
Cell count should be checked by performing a microscopy chamber
count.
✓
A cytospin slide should be prepared, if necessary.
The ‘NRBC Present’ IP message can be switched off in the ‘Analyzer
Settings’ menu. However, if it is switched off but NRBC% exceeds the
threshold value, a ‘WBC Abn Scattergram’ will be triggered instead. Hence,
SEU recommends activating the setting.
(For more information, please contact your local Sysmex representative.)
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‘IG Present’
Suggested actions:
◼ The ‘NRBC Present’ message just alerts the operator of
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
the presence of cells accurately quantitated by the analyser.
IG
◼ Even so, if it is an unknown patient or for the first time, it is
recommended to review a smear to detect other clinically relevant
findings focusing on RBC morphology and report the NRBC results of
the analyser.
IG
IG
Improvements in SW 21.12 onwards
◼ NRBC#/% could be falsely identified in the WNR channel due to preanalytical contamination with lipids, as depicted in the image below.
◼ Lipid particles in the WNR channel are now correctly recognised and
discriminated as ‘ghost particles’ (debris) with the improved algorithm in
SW 21.12.
Details:
◼ XN-Series analysers can report a 6-part DIFF that
comprises NEUT, LYMPH, MONO, EO, BASO and IG; while
in a 5-part DIFF, IG is included in the NEUT count.
WNR scattergram (SFL-FSC)
WNR scattergram (SFL-FSC)
◼ IG includes promyelocytes, myelocytes and metamyelocytes.
◼ The ‘IG Present’ IP message threshold is user-defined and
programmable. It only appears when the IG%/# exceeds the threshold
value.
Suggested actions:
◼ The ‘IG Present’ message alerts the operator to the presence of cells
accurately quantitated by the analyser. The analyser DIFF, including IG
results, can be reported.
SW 00-16
SW 21.12
Parameter
SW 00-16
SW 21.12
NRBC#
0.35 x 103/µL
0.04 x 103/µL (*)
NRBC%
2.5 / 100WBC
0.3 / 100WBC
◼ In case of a 6-part DIFF setting in use, a smear review may be
recommended for an unknown patient to identify the pathology
associated with the cells; in a follow-up for such patients, the reported
IG parameter could be used in monitoring the patient.
◼ A smear review is recommended in the case of the 5-part DIFF setting
in use.
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WBC suspect flags
‘Left Shift?’
WDF scattergram (SSC-SFL)
WDF scattergram (SSC-SFL)
◼ A blood smear review and the manual count are not necessary for
adults, although it would depend on the laboratory standard operating
procedure (SOP).
Details:
◼ The ‘Left Shift?’ flag indicates that the instrument has detected cells in
the region for left shift (band cells) in the WDF scattergram. When band
cells are present, they are included in the neutrophil population.
◼ An increase in band neutrophils typically signals an early response of
white blood cells.
◼ An asterisk [*] appears next to the NEUT#/% and EO#/% (and IG#/%
may be marked).
◼ The flag only occurs if WBC ≧ 0.50 x103/µL.
◼ The asterisk indicates these results are unreliable.
Suggested actions:
◼ In case of neonates, a smear might be indicated. Scan the peripheral
smear for the presence of:
✓
Band cells in increased numbers or immature granulocytes.
✓
Toxic granulation or vacuolation of neutrophils.
✓
Other abnormal cells.
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A brief overview: How does the XN manage the appearance of pathologic
cells in the WDF (and WPC channel)?
In general, the trigger for suspect flags in the WDF and WPC channels is
based on a complex algorithm considering factors such as:
◼ AFLAS is an abbreviation for Adaptive FLagging Algorithm based on
Shape recognition. It is an algorithm that comprehensively analyses the
appearance position of individual clusters (plots) and the shape of the
cluster, and the position of the centre of gravity in the three dimensions,
as indicated in the left image below. This makes it possible to detect
abnormal patterns with high sensitivity.
◼ Forward scatter signals (FSC), side fluorescence signals (SFL), and
side scatter signals (SSC) reflect the functional state of the cells.
◼ Cell counts.
◼ Presence of cells in defined areas (gates) and the ratios of these cell
counts. The right image below depicts the gates that the algorithm
considers while excluding the presence of malignant cells.
Atypical Lymph
◼ The sensitivity of the WDF channel is particularly useful for detecting
inflammations or infections, as well as abnormal WBC populations in
case of malignant diseases.
◼ One benefit of the WDF channel is a presorting of reactive and
malignant diseases by giving the IP messages ‘Atypical Lympho?’ for
reactive diseases or ‘Blasts/Abn Lympho?’ for malignant diseases. In
the latter case, or if both flags are triggered when a differentiation in the
WDF channel is not possible, a further differentiation can be done by
the WPC channel.
The following two sub-sections provide an overview of the two-step
flagging approach between WDF and WPC channels to exclude malignant
samples.
Abnormal Lymph
Blast
Atypical Lymph
Abnormal Lymph
Blast
Variables evaluated by AFLAS
The pathological gates in the
WDF scattergram
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A two-step approach to exclude malignant samples
Exclusion of WBC abnormalities in most samples
Separation of malignant, reactive and negative samples
No flag
No flag
‘Blasts/Abn. Lympho?’
or no flag
(Negative)
(Malignant or negative)
‘Atypical Lympho?'
‘Blasts/Abn Lympho?’ or
‘Atypical Lympho?’
(Reactive)
(Malignant or reactive)
‘Blasts?’ and/or ‘Abn Lympho?’
(Malignant)
‘Blasts?’ and/or ‘Abn Lympho?’
(Malignant)
‘Atypical Lympho?’
(Reactive)
The outcome is one of the more specific suspect IP messages:
◼ ‘Blasts?’ usually points to a myeloid malignancy or lymphoblasts (e.g. in
cases of AML or ALL). Although, in some cases, the presence of
lymphoblasts can trigger ‘Abnormal Lympho?’ instead.
◼ ‘Abnormal Lympho?’ usually points to a malignant (neoplastic) disorder
of lymphocytes (e.g. in cases of chronic lymphatic leukaemia and
lymphomas).
◼ ‘Atypical Lympho?’ usually points to a reactive disorder (e.g. in cases of
infections or inflammations).
◼ Negative when the high specificity of the WPC analysis allows very
reliable detection of WBC abnormalities and further filters out falsepositive samples.
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‘Blasts/Abn Lympho?’
As explained in detail in the brief overview chapter, the WDF channel
performs the initial differentiation of reactive and malignant cells.
‘Blasts/Abn Lympho?’ flag is one of the flags triggered by the WDF channel
when the analyser suspects any malignant conditions. Two example cases
are demonstrated below:
Example case 1 • Chronic Lymphocytic Leukaemia (CLL)
Example case 2 • Acute Myeloid Leukaemia (AML)
Details:
In this example of a diagnosed acute myeloid leukaemia, the ‘Blasts/Abn
Lympho?’ IP message indicates that the instrument has detected abnormal
clustering in the region for blasts in the WDF scattergram.
An asterisk [*] appears next to the NEUT#/%, LYMPH#/%, IG#/% and
MONO#/%. The asterisk indicates these results are unreliable.
Details:
In this example of a diagnosed chronic lymphocytic leukaemia, the
‘Blasts/Abn Lympho?’ IP message indicates that the instrument has
detected significant clustering in the region for abnormal/atypical
lymphocytes located in the upper right area above the lymphocytes in the
WDF scattergram. Due to the low number of events in that region relative
to the lymphocyte cluster, the algorithm cannot exclude malignancy.
Hence, it triggers a ‘Blasts/ Abn Lympho?’ (and ‘Atypical Lympho?) flags.
An asterisk [*] appears next to the NEUT#/%, LYMPH#/%, IG#/% and
MONO#/%. The asterisk indicates these results are unreliable.
In cases of high blast counts, it is common for a ‘Blasts/Abn Lympho?’ IP
message to occur with a ‘WBC Abn Scattergram’ flag since the blasts
overlap with one or more normal cell populations. The analyser will often
detect an abnormal differential.
WDF scattergram (SSC-SFL)
Blasts detected in
the pathological
gates above the
monocyte cluster
WDF scattergram (SSC-SFL)
Abnormal or atypical
lymphocytes detected
Algorithm also detects
the absolute
lymphocytosis reflected
by the dense lymphocyte
cluster
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Suggested actions:
If the WPC channel is available (means XN-20):
‘Blasts?’
The flag triggers an automated reflex measurement for WPC on XN-20
analysers.
Details:
If the WPC channel is not available:
◼ Dashes [----] in place of data:
✓
Perform a manual count and differential according to your lab SOP.
The ‘Blast?’ IP message refers to an analysis in the WPC channel and
indicates significant clustering in the region for blasts.
An asterisk [*] appears next to the NEUT#/%, LYMPH#/%, IG#/% and
MONO#/%. The asterisk indicates these results are unreliable.
◼ Asterisk [*] next to data:
WDF scattergram (SSC-SFL)
✓
Perform a manual differential.
✓
Scan the slide for abnormal cells.
✓
If no abnormalities are found, the data with asterisks [*] can be
reported.
Blasts detected in
the pathological
gates above the
monocyte cluster
◼ Asterisk [*] next to data and ‘greyed-out’ clusters in the scattergram:
✓
Perform a manual differential.
Reflex to WPC
WPC scattergram (SSC-SFL)
Blast gate positive
in the WPC
scattergram
WPC scattergram (SSC-FSC)
Blast gate
positive in the
WPC scattergram
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‘Abn Lympho?’
Details:
The ‘Abn Lympho?’ IP message refers to an analysis in the WPC channel.
It indicates significant clustering in the region for abnormal lymphocytes in
the area with high fluorescence in the WPC scattergram.
An asterisk [*] appears next to the NEUT#/%, LYMPH#/%, IG#/% and
MONO#/%. The asterisk indicates these results are unreliable.
Suggested actions:
◼ When blasts or abnormal lymphocytes are suspected, a smear review
should always be performed to inspect the morphology and count of the
cells.
◼ Perform and report the manual differential results according to your lab
SOP.
WDF scattergram (SSC-SFL)
Abnormal lymphocytes
detected together with an
absolute lymphocytosis
Reflex to WPC
WPC scattergram (SSC-SFL)
Abnormal lymphocytes
detected
in the WPC scattergram
WPC scattergram (SSC-FSC)
The visible change in
the distribution of
lymphocytes between
the two clusters was
also detected by the
flagging algorithm
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Blasts AND Abnormal Lympho?
Details:
The ‘Blasts?’ AND ‘Abnormal Lympho?’ IP messages refer to an analysis
in the WPC measurement channel and indicate significant clustering in the
region for blasts and abnormal lymphocytes. This can occur in samples
with circulating blasts and detectable abnormal lymphocytes, such as the
example below.
The above scattergrams belong to an acute myeloid leukaemia sample.
The algorithm detects the abnormal lymphocyte and monocyte clouds in
WDF and consequently triggers ‘Blasts?’ AND ‘Abn Lympho’ flags after the
WPC measurement.
An asterisk [*] appears next to the NEUT#/%, LYMPH#/%, IG#/% and
MONO#/%. The asterisk indicates these results are unreliable.
WNR scattergram (SFL-FSC)
Suggested actions:
◼ When blasts or abnormal lymphocytes are suspected, a smear review
should always be performed to inspect the morphology and count of the
cells.
WDF scattergram (SSC-SFL)
The algorithm detects
abnormalities in the
lymphocyte and
monocyte clouds. Thus,
triggering a check in the
pathological gates for
both, abnormal
lymphocytes, and
abnormal monocytes
WPC scattergram (SSC-FSC)
Positive blast
gate in the WPC
scattergram
◼ Perform and report the manual differential results according to your
laboratory SOP.
WPC scattergram (SSC-SFL)
Abnormal lymphocytes
detected
in the WPC scattergrams.
Interference from NRBC can
be ruled out based on the
WNR scattergram.
Positive blast
gate in the
WPC
scattergram
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‘Atypical Lympho?’
Improvements with SW 21.12
Details:
The algorithm was improved to provide more specific flagging in samples
with clinically significant atypical (reactive) cells concentrations.
✓ The ‘Atypical Lympho?’ Q-flag correlates with the percent of reactive
lymphocytes (RE-LYMP%).
The ‘Atypical Lympho?’ IP message indicates that the instrument has
detected significant clustering in the region for atypical lymphocytes
(located in the upper right lymphocyte region on the WDF scattergram) or
combined with the WPC channel after a malignancy has been excluded.
An asterisk [*] appears next to the NEUT#/%, LYMPH#/%, IG#/% and
MONO#/%. The asterisk indicates these results are unreliable.
The lymphocyte population has a variable presentation at different stages
of infective processes. This can be seen in the scatterplots below.
✓
A Q-flag of 100 equals 6% of RE-LYMP and triggers the ‘Atypical
Lympho?’ flag as per the default setting.
✓
By modifying the Q-flag value, the laboratory can decide which RELYMP% value triggers the flag.
The table below summarises the correlation between the RE-LYMP% and
Q-flag value:
Q-flag value
RE-LYMP%
10
0.5 – 1.1%
20
1.2 – 1.7%
30
1.8 – 2.3%
40
2.4 – 2.9%
50
3 – 3.5%
60
3.6 – 4.1%
70
4.2 – 4.7%
Suggested actions:
80
4.8 – 5.3%
◼ Dashes [----] in place of data:
90
5.4 – 5.9%
Active phase
✓
Recovery phase
Normal
Perform a manual count and differential according to your lab SOP.
◼ Asterisk [*] next to data:
100
6 – 6.8%
DEFAULT
110
6.9 – 7.7%
✓
Perform a manual differential.
120
7.8 – 8.6%
✓
Scan the slide for abnormal cells.
130
8.7 – 9.5%
✓
If no abnormalities are found, the data with asterisks [*] can be
reported.
140
9.6 – 10.4%
300
20%
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5
RBC abnormal flags
RBC flagging
‘RBC Abn Distribution’
RBC histogram from RBC/PLT channel
◼ The RBC histogram plots cell volume (x-axis) and frequency (y-axis).
Normal samples display a Gaussian curve.
◼ The small peak to the left of the main peak in the RBC is the PLT
histogram (discussed later). Information about possible interferences
can be gained by reviewing these histograms.
Details:
The ‘RBC Abn Distribution’ IP message is generated when the histogram
pattern is abnormal or RBC < 0.5 x 106/µL.
RBC histogram
RBC histogram
Abnormal height at lower discriminator (LD)
PLT
RBC histogram
RBC histogram
Small RBCs
Large RBCs
◼ The image below depicts the parameters derived from the RBC
histogram, several of which are used as judgement criteria to exclude
anomalies.
Two RBC histograms with multiple peaks
RBC histogram
Frequency
100%
•
•
68.26% of RBC
histogram (RDW-CV)
◼ Dashes [----] appear in place of data that could not be calculated, or
certain parameters may be marked with an asterisk [*]. The asterisk [*]
indicates these results are unreliable.
◼ The different triggers for ‘RBC Abn Distribution’ are described more in
detail on the following pages.
•
•
LD
RDW-SD
(25-75 fL)
20%
Cell volume
UD
(200-250 fL)
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‘Dimorphic Population’
Example case • Chronic Lymphocytic Leukaemia (CLL)
Details:
The ‘Dimorphic Population’ IP message is generated when there are
multiple peaks in the RBC histogram pattern.
RBC histogram
RBC histogram
In this case, the small lymphocytes due to the massive
leukocytosis interfere with the RBC count, as seen in the histogram.
Hence, the RBC count is falsely elevated. Only the first peak would
comprise the RBC, whereas the second peak is the WBC.
WDF scattergram (SSC-SFL)
WNR scattergram (SFL-FSC)
RBC histogram
◼ Dashes [----] appear in place of data for the RDW-SD and RDW-CV.
◼ This message may cause certain RBC parameters to be marked with
an asterisk [*]. The asterisk [*] indicates these results are unreliable.
Suggested actions:
◼ Scanning the peripheral smear for the presence of abnormal RBC
morphology such as:
✓
marked anisocytosis
✓
multiple RBC populations
✓
RBC fragments
✓
poikilocytosis
✓
rouleaux or RBC agglutination (refer to suggested action for ‘RBC
Agglutination?’ if present)
◼ If no abnormalities are found, the results with the asterisk [*] may be
reported.
Suggested actions:
◼ The RBC count and MCV for the two visible populations in the RBC
histogram can be found in the Service -> RBC/PLT tab of the Browser
Screen if needed.
◼ R-MFV refers to the most frequent volume of RBC. The mean MCV and
mode (the most frequent volume) are equal in a normal Gaussian
distribution. However, in case of outliers caused by agglutination, the
mean and mode are shifted; R-MFV = mode in such a curve
representing the RBC population.
◼ R-MFV value can be used to manually correct the MCH, HCT and
MCHC.
NOTE: These results are derived from the Service Tab. The decision to
report these parameters is the user's responsibility.
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RBC suspect flags
‘Turbidity/HGB Interf?
Details:
◼ The ‘Turbidity/HGB Interf?’ IP message occurs when the MCHC is
greater than 36.5 g/dL (22.7 mmol/L) due to interference either in the
HGB measurement channel or in the RBC/PLT measurement channel;
in some cases, it is a true increase in MCHC.
◼ Asterisks [*] appear next to the HGB, MCH and MCHC parameters. The
asterisk indicates these results are unreliable.
↑ MCHC =
HGB
HCT
↑
↓
RBC Agglutination?’
Details:
◼ The ‘RBC Agglutination?’ flag is triggered when the MCHC is greater
than 40.0 g/dL (24.8 mmol/L), and certain parameters such as RBC,
MCH and the upper detection limit of the RBC histogram exceed index
values due to interference either in the HGB measurement channel or
RBC/PLT measurement channel.
◼ Asterisks [*] appear next to the RBC, RET#, HCT, MCV, MCH and
MCHC parameters. The asterisk indicates these results are unreliable.
RBC histogram
Interference detected
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31
Possible causes:
Suggested actions:
The laboratory’s challenge is to determine the cause of the increased MCHC and to take appropriate corrective action. The CBC-O concept
embedded in the Extended IPU helps to resolve the problem caused by the abovementioned interferences in traditional measurement
methodologies and automatically offers the proper corrective actions using RET channel technology (RBC-O and HGB-O). This ensures an
optimal CBC result is reported for every sample. (For more information please contact your local Sysmex representative.)
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32
‘Iron Deficiency?’
‘HGB Defect?’
Details:
Details:
◼ The ‘Iron Deficiency?’ IP message is determined by calculation and size
comparison of certain RBC parameters (MCHC, MCV, RDW-CV).
◼ The ‘HGB Defect?’ IP message is determined by calculation and size
comparison of certain RBC parameters (MCV and RDW-CV).
◼ The flag only occurs if RBC ≧ 0.50 x 10⁶/µL.
◼ The flag only occurs if RBC ≧ 0.50 x 10⁶/µL.
◼ The presence of this flag triggers no asterisk marks.
◼ The presence of this flag triggers no asterisk marks.
◼ Results are reliable.
◼ Results are reliable.
RBC histogram
RBC histogram
Suggested actions:
Suggested actions:
Follow-up according to your laboratory SOP for such patients.
Follow-up according to your laboratory SOP for such patients.
◼ Actions may include
◼ Actions may include scanning the peripheral smear for the presence of
abnormal RBC morphology.
✓
Measure RET channel for RET-He.
✓
Scan the peripheral smear for the presence of abnormal RBC
morphology.
◼ Report the presence of any clinically significant RBC morphology
abnormalities according to your laboratory SOP.
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‘iRBC?’
‘iRBC?’ flag - Correcting the WBC count and differential for RBC with
inclusions
◼ RBC with inclusions, e.g. parasites, might react with the reagents and
acquire fluorescence or have an increased side scatter and interfere
with the WBC in the scattergrams.
Example case • A positive sample for Plasmodium
vivax.
The right panel below shows the WNR scattergram of the same sample
with the activated ‘iRBC?’ flag.
WNR scattergram (SFL-FSC)
WNR scattergram (SFL-FSC)
◼ This interference can lead to the analyser reporting false results, such
as a falsely increased WBC count from the WDF channel (WBC-D), as
well as a misclassified differential.
◼ Even a small number of inclusion RBC may have a significant impact on
the WBC count, as the concentration of RBC is a thousand-fold higher
than WBC.
◼ To solve this problem, Sysmex has developed the ‘iRBC?’ flag,
triggered when the analyser detects the presence of such RBC that
influence the WBC-D count and differential.
‘iRBC?’ NOT activated
‘iRBC?’ activated
‘iRBC?’ algorithm in the WDF channel
Details:
In XN-Series, the RBC with inclusions can be detected in the WNR and
WDF channels. Hence, the ‘iRBC?’ flag can be triggered by both channels.
‘iRBC?’ algorithm in the WNR channel
◼ In the WNR channel, the RBC with inclusions do not overlap with the
WBC area. Thus the WBC count from WNR (WBC-N) is not influenced
by the infected RBC.
◼ When the ‘iRBC?’ licence is activated, the RBC with inclusions are
clearly indicated by a dark purple colour, as shown in the figure below.
◼ In the WDF channel, inclusions in RBC can interfere with the WBC-D
and neutrophil/eosinophil cell counts.
◼ When the ‘iRBC?’ licence is activated, the analyser uses an algorithm
including FSC to separate white blood cells from the inclusion RBC, as
the interference can be clearly seen in the WDF (SSC–FSC)
scattergram. Also, in this scattergram, the interference is clearly
indicated by a purple colour representing the inclusion RBC.
◼ Consequently, the WBC-D count and the differential are automatically
corrected, indicated by an ‘&’.
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6
Example case • A sample positive for Plasmodium
vivax.
The right panel below shows the WDF scattergram of the same sample
with the activated ‘iRBC?’ flag.
RET flagging
RET channel scattergrams
The normal distribution of the populations in RET channel
scattergrams for a healthy person is shown below:
RET scattergram (SFL-FSC)
WDF scattergram (SSC-SFL)
‘iRBC?’ NOT activated
WDF scattergram (SSC-SFL)
‘iRBC?’ activated
Suggested action:
RET abnormal flag
◼ Although the WBC counts and WBC differential are correct, a smear
review might be useful to investigate the cause behind the ‘iRBC?’ flag.
‘RET Abn Scattergram’
✓
PLT-O scattergram (SFL-FSC)
Scan slide for abnormal cells (RBC abnormalities, parasites etc.).
(For more information on the ‘iRBC?’ flag, please contact your local
Sysmex representative.)
Details:
◼ The ‘RET Abn Scattergram’ IP message indicates that the analyser has
detected an abnormal separation between the RBC and reticulocytes at
RET_THR (threshold) main RET scattergram or increased activity in the
RET_UPP (RET Upper Particle Plateau) area on the RET(EXT)
scattergram.
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◼ In the RET (EXT) Scattergram, within the RET-UPP area (green particle
area past reticulocytes), NRBCs, Howell-Jolly Bodies, parasites, or
stress reticulocytes can appear. These are typically not included in the
reticulocyte count.
RET scattergram
RET_THR
RET scattergram
RET scattergram
RET (EXT) scattergram
RET_UPP
P
Malaria – P. falciparum
RET scattergram
Basophilic stippling due to a
genetic haemoglobin mutation
RET (EXT) scattergram
◼ ‘RET Abn Scattergram’ can also be triggered due to anomalies related
to the PLT-O scattergram, such as interference by small leucocytes or if
there is no clear separation between the RBC/RET and PLT-O clusters.
PLT-O scattergram
PLT-O scattergram
Spherocytes
RET scattergram
Detection area for small
leukocytes
Small lymphocytes interfering
with the RET-UPP area
RET (EXT) scattergram
No clear separation between
RBC/RET and PLT-O clusters
◼ Depending on the reason for the flag, the RET%, RET#, IRF and PLT-O
parameters can be marked with an asterisk [*]. The asterisk indicates
these results are unreliable.
◼ Some examples of interferences that can trigger ‘RET Abn
Scattergram’ are as follows;
Heinz bodies
NRBC interference in the
RET-UPP area
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Suggested actions:
◼ Review the peripheral blood smear for the presence of NRBC, HowellJolly bodies, spherocytes, basophilic stippling etc.
Suggested actions:
◼ Scan the peripheral smear for the presence of RBC
fragments and other poikilocytosis.
◼ If present, report the results with a comment stating that the reticulocyte
results may be affected by these interfering substances.
◼ Report the presence of any clinically significant RBC morphology
according to individual laboratory SOP.
◼ Or perform the reticulocyte count as an alternative method.
◼ Check the PLT-I count for possible interferences.
◼ Decisions to report with a comment or perform an alternative method
should be based on your laboratory SOP.
RET suspect flags
‘Fragments?’
Details:
◼ The ‘Fragments?’ IP message can be triggered by the RET scattergram
and/or the RBC histogram (from the RBC /PLT channel).
◼ The algorithm considers certain RBC and PLT parameters (MCV, RDWSD, MCHC, PLT, lower RBC discriminator*, upper PLT discriminator*)
* These are not reportable parameters but are used in the internal flagging algorithm.
RET scattergram
Lower RBC discriminator
Upper PLT discriminator
The approximate area where the
algorithm considers the presence of
fragments
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7
PLT abnormal flags
PLT flagging
PLT histogram from RBC/PLT channel
‘PLT Abn Distribution’
The image above shows a PLT histogram with normal platelet distribution.
It depicts the parameters derived from the PLT histogram, several of which
are used as judgement criteria to exclude anomalies.
◼ The ‘PLT Abn Distribution’ IP message is triggered when certain
parameters exceed defined thresholds or cannot be analysed (PDW,
PLT-I, P-LCR, MPV, lower RBC histogram discriminator*, platelet upper
discriminator*).
PLT histogram
Frequency
100%
◼ Dashes [----] may appear in place of data for the PDW, MPV, P-LCR
and PCT or they are marked with an asterisk [*], which indicates the
results may be unreliable.
◼ The reasons for an abnormal platelet histogram distribution are several,
some of which are discussed below.
Suggested actions:
P-LCR
PDW
LD
(2-6 fL)
12 fL
20%
UD Cell volume
(12-30 fL)
PLT-F channel scattergram
PLT-F scattergram (SFL-FSC)
PLT histogram
Type ‘A’ curve: Abnormal height at the upper
discriminator (PU).
PLT-I result is still reliable, as the areas on both
sides are almost identical.
Type ‘B’ curve’: Abnormal height at the upper
discriminator (PU).
PLT-I result is not reliable and should be checked
with an alternative PLT measurement method.
Such curves are typically caused due to an
interference by RBC fragments and microcytes.
Type ‘C’ curve: Abnormal height at the upper
discriminator (PU).
PLT-I result is not reliable and should be checked
with an alternative PLT measurement method.
Such curves are typically caused by in
interference due to giant platelets and/or PLT
aggregates.
*These are not reportable parameters but are used in the internal flagging algorithm.
Normal distribution of the populations in PLT-F
scattergram for healthy person is shown.
38
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‘PLT Abn Scattergram’
If the analyser triggered ‘PLT Abn Distribution’ and the PLT count is
marked unreliable [*]:
◼ No RET and no PLT-F available - PLT review on the slide.
◼ Scan the peripheral smear to review for the presence of abnormal RBC
or PLT morphology, such as:
✓
Large or giant platelets
✓
Small platelets
✓
Platelet clumps
✓
RBC fragments
✓
Microcytic RBC
◼ The PLT-F channel only triggers the ‘PLT Abn Scattergram’ flag on the
XN-Series analyser.
◼ This IP Message occurs when clustering in the platelet and IPF area in
the PLT-F scattergram is abnormal.
◼ PLT-F, IPF# and IPF% will be marked with an [*], which indicates these
results are unreliable.
PLT-F scattergram (SFL-FSC)
◼ Report according to your laboratory SOP if abnormal RBC, PLT, or
other morphology is noted.
◼ If no PLT-F licence channel is available, but the RET channel is
activated, measure the RET channel for PLT-O. In the absence of other
PLT-related IP messages and a reliable result, the PLT-O may be
reported with no further action.
◼ If an active PLT-F channel is available, measure PLT-F. The PLT-F
may be reported with no further action in the absence of other PLTrelated IP messages and a reliable result.
‘PLT Abn Scattergram’ was triggered
due to an indistinguishable border
(indicated with red dotted line) between
debris and the PLT-F cluster.
NOTE: Unlike the X-Class analysers, where PLT abnormalities in the RET
channel triggered ‘PLT Abn Scattergram’, for XN, abnormalities in the PLTO scattergram will trigger ‘RET Abn Scattergram’ (as explained earlier)
because the ‘PLT Abn Scattergram’ IP message is exclusive for PLT-F
channel.
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PLT suspect flags
Suggested actions:
◼ Review results according to your laboratory SOP. This may include
scanning the peripheral smear to review for the presence of abnormal
RBC or PLT morphology, such as:
✓
Large or giant platelets
✓
Platelet clumps
✓
RBC fragments
✓
Microcytic RBC
◼ If abnormal RBC, PLT or other morphology is noted, report according to
the laboratory SOP.
NOTE: Reviewing the feathered edge and sides of the peripheral smear is
suggested as platelet clumps, and fibrin strands may migrate to this area
during smear preparation.
‘PLT Clumps?’
Details:
◼ The ‘PLT Clumps?’ flagging strategy is based on areas in the WNR,
WDF and/or PLT-F channels. The algorithm also considers parameters
such as MicroR%, P-LCR and the PLT count.
◼ It detects abnormalities like an increased FSCW in the WNR, WDF and
PLT-F (FSCW-FSC) plots, respectively. This is of value since particles
with an increased FSCW are normally PLT clumps.
◼ From SW 21.12, the algorithm uses the information from the FSCWFSC scattergrams also to exclude the likely presence of giant platelets.
The areas where the algorithm checks for probable PLT clumps on WNR,
WDF and PLT-F scattergrams are indicated below:
WNR scattergram (SFL-FSC)
WNR scattergram (SFL-SSC)
WNR scattergram (FSCW-FSC)
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WDF scattergram (FSCW-FSC)
PLT-F scattergram (FSCW-FSC)
Suggested actions:
◼ Follow your laboratory SOP. Possible actions may include:
◼ Checking the sample for the presence of clumps.
◼ Scanning the peripheral smear, especially the feathered edge, for the
presence of abnormal morphology, including:
✓
Fibrin strands
✓
Platelet clumps
✓
If there is still a ‘PLT Clumps?’ IP message and platelet clumps are
present on the smear review. It could be an in vitro reaction with
EDTA. Analyse the sodium citrate tube. Obtain only PLT counts from
the sodium citrate tube, as sodium citrate alters RBC morphology
and indices.
✓
Multiply PLT results from the sodium citrate tube by the factor used
by your laboratory.
✓
If recollection is not possible or platelet clumps persist when using
sodium citrate, estimate the platelet count and report it as decreased,
adequate or increased and comment on the platelet clumps
according to your laboratory SOP.
NOTE: Sodium citrate tubes are not specified in the Instructions for Use
and consequently out of specifications. The user must validate tube
suitability to be used in routine.
NOTE: Reviewing the feathered edge and sides of the peripheral smear is
suggested as platelet clumps, and fibrin strands may migrate to this area
during smear preparation.
◼ If platelet clumps or fibrin strands have interfered, perform one of the
following alternate procedures to obtain an accurate count:
✓
Re-draw specimen in EDTA and sodium citrate tubes if possible.
Analyse re-drawn EDTA tube. If the repeat run has no ‘PLT Clumps?’
IP message, report these results.
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‘Giant Platelet?’
Details:
Suggested actions:
◼ The ‘Giant Platelet?’ flag was implemented with SW 21.12 onwards and
is part of the ‘PLT Clumps?’ flagging strategy.
◼ Follow your laboratory SOP.
◼ It enables the XN analyser to distinguish them from platelet clumps,
reducing the false positive ‘PLT Clumps?’ flags by indicating the
possible presence of abnormally large platelets in the sample.
◼ From SW 22.06 onwards, if the sample is positive for ‘Giant Platelet?’
an asterisk [*] appears next to the PLT, PDW, MPV, P-LCR and PCT,
which indicates the results are unreliable.
◼ The WNR channel can only trigger the ‘Giant Platelet?’ flag. In the PLTF channel, an increased IPF% and IPF# indicate the presence of giant
and reticulated platelets.
WNR scattergram (SFL-SSC)
PLT clumps
◼ If a slide review is performed and abnormalities are detected, report
results with a related comment as per your laboratory SOP.
NOTE: The availability of this flag depends on your system configuration!
Additional information:
Based on the channel priority, the different likely outcomes of the ‘PLT
clumps’ strategy (with an activated ‘Giant Platelet?’) are summarised in the
matrix below:
WNR scattergram (FSCW-FSC)
Giant PLT
PLT clumps
The analyser uses the information from the WNR (SFL-SSC) and WNR (FSCWFSC) scattergrams to distinguish between giant platelets and PLT clumps.
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8
Action messages
The XN analyser may trigger an ‘Action Message’ to indicate to the user a likely follow-up action due to an atypical result caused by interference or, for
example, a sample mix-up. There are three main types of ‘Action Messages’:
◼ Check the sample. [Check] - There may be a mix-up of samples. Otherwise, there is a significant difference in the analysis results.
◼ [Review] - Channel difference has occurred. Check the analysis results
◼ [Retest] - Check the analysis mode, the order and the status of the sample, and then reanalyse.
The table below provides an overview of the different action messages, the underlying trigger and possible follow-up actions.
Action
Displayed message
Underlying trigger conditions
Suggested Actions
[Check]
The sample might be
wrong. Check the
sample.
Delta check is related to a previous
measurement of the same patient. The trigger
includes a floating threshold based on the
current value of certain parameters.
Check the sample.
[Check]
A significant change in
HGB. Check the
sample.
Delta check is related to a previous
measurement of the same patient. The trigger
includes a floating threshold based on the
current value of certain parameters.
Check the sample.
[Check]
A significant change in
MCV. Check the
sample.
Delta check is related to a previous
measurement of the same patient. The trigger
includes a floating threshold based on the
current value of certain parameters.
Check the sample.
[Review]
Difference between
WNR and WDF.
Check the results.
It is generated based on the ratio of the Total
Nucleated Count in the WDF channel (TNC-D)
to the Total Nucleated Count in the WNR
Channel (TNC-N). The ratio is calculated as
TNC-D / TNC-N.
◼
◼
NOTE: If the analyser has reported the WBC
from the WDF channel, the WBC result will
have the ‘&D’ indicator appended to the
parameter.
Channels
involved
Rerun the sample.
If the message is not eliminated, verify WBC and differential results
according to your laboratory SOP. Possible actions may include:
✓ Scanning the slide for abnormal cells and estimating the WBC count.
✓ Performing a manual differential if abnormal cells are observed.
✓ If no abnormalities are found when reviewing the smear, and the WBC
estimate matches the analyser reported WBC, the results may be
reported according to your laboratory SOP.
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[Review]
Difference between
RBC and RET.
Check the results.
It is generated based on the ratio of the RBC
result from the RET channel (RBC-O) and the
RBC result from the impedance channel.
The ratio is calculated as RBC-O / RBC.
◼
◼
◼
[Review]
Aged sample?
◼
Depending on the individual workflow challenges (created by aged
samples) for each lab, the ASI may or may not be needed. If needed, it will
have to be activated by a Sysmex representative after obtaining a license
agreement.
In case of a sample flagged as ‘Aged Sample?’, follow the laboratory SOP.
Possible actions could include:
✓ If only ‘Aged Sample?’ is triggered, no smear is necessary. Reanalyse using a fresh sample.
✓ ‘Aged Sample? ` triggered along with a pathological flag ‘Blasts/ Abn
Lympho?’ with or without ‘Atypical Lympho?’, smear review is
necessary.
◼
◼
Aged samples are described most
prominently by an increase in MCV, a
decrease in MCHC and changes in WDF
cloud shape and position.
These changes are not exclusive to aged
samples but occur in certain pathological
samples.
The ‘Aged Sample Identifier’ (ASI) can
differentiate between aged, pathological,
and pathological samples.
Rerun the sample.
If the message is not eliminated, follow your laboratory SOP.
Possible actions may include:
✓ Scanning the peripheral smear for the presence of abnormal RBC
morphology such as rouleaux or polychromasia, parasites.
✓ Verify the reticulocyte count using an alternative method.
[Review]
Difference between
PLT and PLT-F.
Check the results.
It is generated based on the ratio of the PLT
result from the PLT-F channel and the PLT
result from the impedance channel (PLT-I).
PLT-F results are always significantly higher
compared to PLT-I. The ratio is calculated as:
PLT-F / PLT-I.
◼
◼
Rerun the sample.
If the message is not eliminated, follow your laboratory protocol to count
PLT using an alternative method.
[Retest]
Reflex PLT.
PLT measured by the impedance
measurement method might be interfered with
by fragmented RBC, microcytes or giant
platelets leading to an unreliable PLT-I result.
◼
Reflex PLT-O or PLT-F. If not available, follow the laboratory protocol.
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[Retest]
Suspect sample,
check the sample.
Insufficient sample mixing (caused by the
presence of interference such as proteins or
hyaluronic acid).
The interference is mostly visible in the WNR
(SFL-SSC scattergram).
◼
◼
◼
Remix and rerun the sample.
If the message is not eliminated, follow your laboratory SOP.
If the initial and repeat run results are consistent and consistent with
patient history, report according to your laboratory SOP.
◼
If the results from the initial and repeat runs are consistent, there is no
previous patient history, and the results are abnormal, confirm as
required by your laboratory SOP using smear review or an alternate
method.
If the results from the initial and repeat runs are NOT consistent, consider
insufficient or non-mixing in manual mode, an overfilled tube (e.g., no air
space in the tube to enhance hand or automated mixing) or a clotted or
fibrinous sample. Reject or recollect the sample based on your laboratory
SOP.
If there are any flags or unreliable results on the repeat run, follow your
laboratory SOP for that flag.
WNR scattergram (SFL-SSC)
◼
◼
[Retest]
Suspect sample,
check the sample.
With SW 22.06 onwards, a new algorithm has
been implemented to detect falsely high WBCN caused by unknown interference.
◼
◼
Rerun the sample.
It is also possible to repeat the sample using a CBC+DIFF profile.
[Retest]
Confirm eosinophil
and neutrophil count
by other methods
From SW 22.08 onwards, when the algorithm
is unsure of the NEUT/EO clusters in case of a
difficult classification, it will trigger a ‘WBC Abn
Scattergram’ message and this action
message. Both these messages are subject to
‘Service Settings’. Please speak to your local
Sysmex representative for more details.
◼
Review the neutrophils and eosinophils in a smear review.
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9
Possible sample interferences
Some abnormal samples may interfere with automated cell counting methods. The following is a list from the XN-Series Instructions for Use of possible
substances that may interfere with the listed parameters.
Cell type
Affected parameter
Falsely low WBC count
White blood cells (WBC)
Possible interference
White blood cell aggregation
Falsely high WBC count
◼
◼
◼
Possibility of PLT clumps
Cryoprotein
Cryoglobulins
◼
◼
Fibrin
Giant platelets
Falsely low RBC count
◼
◼
Red blood cell aggregation (cold agglutination)
Microcytic red blood cells
◼
Possibility of RBC fragments
Falsely high RBC count
◼
Leucocytosis (> 100,000/μL)
◼
Giant platelets
Falsely high HGB count
◼
◼
Leucocytosis (> 100,000/μL)
Lipaemia
◼
Abnormal protein
Falsely low HCT count
◼
◼
Red blood cell aggregation (cold agglutination)
Microcytic red blood cells
◼
Possibility of RBC fragments
Falsely high HCT count
◼
◼
Leucocytosis (> 100,000/μL)
Severe diabetes
◼
◼
Uraemia
Spherocytosis
Falsely low PLT count
◼
◼
Possibility of PLT clumps
Pseudothrombocytopenia
◼
Giant platelets
Falsely high PLT count
◼
◼
◼
Microcytic red blood cells
Possibility of RBC fragments
White blood cell fragments
◼
◼
Cryoprotein
Cryoglobulins
Falsely high RET count
◼
◼
◼
Severe red blood cell aggregation (cold agglutination)
Giant platelets
Possibility of PLT clumps
◼
◼
◼
White blood cell fragments
Malaria
Howell-Jolly body
Red blood cells (RBC)
Haemoglobin (HGB)
Haematocrit (HCT)
Platelets (PLT)
Reticulocytes (RET)
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46
WBC-BF
10
Falsely high WBC
◼
◼
◼
◼
Liposome preparation
Excessive mixing of the sample
Debris
Fat globule
◼
◼
◼
◼
Crystals
High viscosity synovial fluid
Bacteria
Fungi
Supporting literature
◼ Genevieve F et al. (2014): Smear microscopy revision: propositions by the GFHC, Feuillets de Biologie (Vol LVI N° 317). Link:
http://www.gfhc.fr/fr/documents/theme-3-recommandations
◼ Briggs C et al. (2012): Performance evaluation of the Sysmex haematology XN modular system. J Clin Pathol 65: 1024-30 Link:
http://dx.doi.org/10.1136/jclinpath-2012-200930 (abstract available from Sysmex upon request)
◼ Kawauchi S et al. (2013): The positions of normal leukocytes on the scattergram of the newly developed abnormal cell detection channel of the XNseries multi-parameter automated hematology analysers. Sysmex J Int 23(1): 1-9. Link: Free online (after free registration)http://scientific.sysmex.co.jp/en/
◼ Berda-Haddad Y et al. (2016): Increased mean corpuscular haemoglobin concentration: artefact or pathological condition? Int J Lab Hematol
39(1):32-41 Link: https://onlinelibrary.wiley.com/doi/epdf/10.1111/ijlh.12565
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