Clin Chem Lab Med 2017; aop
Letter to the Editor
Michela Seghezzi*, Barbara Manenti, Giulia Previtali, Andrea Gianatti, Paola Dominoni
and Sabrina Buoro
A specific abnormal scattergram of peripheral
blood leukocytes that may suggest hairy cell
leukemia
https://doi.org/10.1515/cclm-2017-0763
Received August 26, 2017; accepted October 31, 2017
Keywords: CBC; cell population data; hairy cell leukemia;
WDF scattergram; WPC channel; XN-module.
To the Editor,
The hematology analyzer (HA) Sysmex XN-9000 (Sysmex,
Kobe, Japan) (XN-module) is equipped with novel tech-
nologies for white blood cell (WBC) count and differen-
tial, represented by its three specific channels, namely,
WBC differential (WDF), white progenitor cell (WPC) and
white cell nucleated (WNR) channel. The WDF comes also
with a new set of parameters added to the traditional leu-
kocyte differential that provides further qualitative data
for peripheral blood (PB) interpretation, like abnormal
scattergram and cell population data (CPD) [1]. Further-
more, the ‘blasts’ and ‘abnormal lymphocytes’ flags can
be shown in the WPC channel. This morphological flags,
combined with the information coming from the WNR and
WDF scattergrams (i.e. the traditional scattergram ren-
dering of the WBC population), can often represent the
instrumental counterpart of a malignant alteration in leu-
kocyte morphology [2], thus offering a powerful warning
indicator in detecting hemopathologies through easy-to-
measure and reproducible analytical variations in the PB
by HAs [3].
*Corresponding author: Michela Seghezzi, Clinical Chemistry
Laboratory, Hospital Papa Giovanni XXIII, Piazza OMS 1, 24127
Bergamo, Italy, Phone: (+0039) 0352674550,
Fax: (+0039) 0352674939, E-mail: m.seghezzi89@gmail.com
Barbara Manenti, Giulia Previtali, Paola Dominoni and Sabrina
Buoro: Clinical Chemistry Laboratory, Papa Giovanni XXIII Hospital,
Bergamo, Italy
Andrea Gianatti: Pathology Laboratory, Papa Giovanni XXIII
Hospital, Bergamo, Italy
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In this case report, we describe our experience with
the XN-module with the WDF, WNR and WPC channels in
investigating a case that was later diagnosed as hairy cell
leukemia (HCL) by immunophenotypic and cytogenetic
analysis.
A 65-year-old male came to the attention of our Hos-
pital Dermatology Service after beach holiday for the
treatment of a suspect urticaria. The patient presented
cutaneous manifestations consisting of pruritic papules
on the trunk and extremities, together with periorbital
swelling of both eyes with a normal conjunctiva and
fever.
A PB sample was tested with a full serum biochemis-
try panel and a complete blood count (CBC); the latter was
performed on the XN-module (results shown in Table 1).
The CBC revealed a trilinear pancytopenia without any
morphological flags nor in WDF channel nor in WPC
channel performed as reflex test (chosen by operator) as
the WDF channel showed an abnormal scattergram.
The WNR, WDF and WPC scattergrams were all sig-
nificantly distinct from those of a healthy adult subject
(Figure 1A–C), exhibiting in this case some peculiar alter-
ations, consisting of the following: (a) WDF (Figure 1F),
the presence of an abnormal monocyte cluster (colored in
green), clearly lower than the usual position (as pointed
by the black arrow); (b) WNR (Figure 1G), an abnormal
lymphocyte double cluster (colored in pink and indicated
by the two arrows); and (c) WPC (Figure 1H), the complete
lack of the monocyte cluster (i.e. the upper white area
pointed out). WNR and WPC scattergrams showed the
complete lack of monocyte cluster (i.e. the upper white
area pointed out by arrow indicate the zone in which it
should be visible). In the opposite way in the WDF scat-
tergram, monocyte abnormal cluster was present in
association with abnormal lymphocytes cluster. Due to
the presence of both pancytopenia, and abnormal scat-
tergrams, a PB smear review by optical microscopy (OM)
was performed. PB smear was prepared by SP-10 auto-
mated slide maker (Sysmex, Kobe, Japan), stained with
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2      Seghezzi et al.: WDF scattergram on suspect of hairy cell leukemia

Table 1: Data of 65-year-old patient with hairy cell leukemia (HCL) and adult patient with B-cell marginal splenic marginal zone lymphoma
(SMZL).

HCL (onset) SMZL Reference range

White blood cells (×10 /L)

9
3.08 4.99 4.20–9.40
Hemoglobin, g/L 90 134 142–172
Red blood cells (×1012/L) 3.05 4.46 4.70–5.82
RBC mean corpuscular volume, fL 90.8 87.2 81.8–95.3
Platelet (×109/L) 95 72 150–400
Neutrophils (×109/L) 1.09 2.01 2.00–6.70
Lymphocytes (×109/L) 1.71 2.70 1.13–3.40
Monocytes (×109/L) 0.26 0.21 0.25–0.80
Eosinophils (×109/L) 0.01 0.04 0.00–0.50
Basophils (×109/L) 0.00 0.03 0.00–0.10
Morphology flags Absent Atypical Lympho? Absent

Hematological parameters (including differential leukocyte).

A B C D E

F G H I L

M N O P Q

Figure 1: Sysmex XN-module scattergrams performed by white cell differential (WDF), white progenitor cell (WPC) and white cell nucleated
(WNR) channels and peripheral blood (PB) microphotographs (May-Grunwald Giemsa stain at 1000 ×) by Sysmex DI-60 on PB of a healthy
adult subject (from A to E), a 65-year-old patient with hairy cell leukemia (HCL: from F to L) and an adult patient with B-cell splenic marginal
zone lymphoma (SMZL: from M to Q).
Healthy adult subject: (A) WDF scattergram: the different leukocyte classes are represented by different color clusters as follows: lympho-
cytes (pink), monocytes (green), neutrophils (light blue) and eosinophils (red). Normal distribution of WBC clusters; (B) WNR scattergram:
regular pattern; (C) WPC scattergram: regular pattern; (D) a normal monocyte; (E) a normal lymphocyte, HCL: (F) WDF scattergram: an
abnormal monocyte cluster (as pointed by the arrow) spreads over the lymphocyte zone; (G) WNR scattergram: it is clearly evident the
presence of an abnormal lymphocyte double-cluster (indicated by the two arrows) and the lack of monocyte cluster; (H) WPC scattergram: a
complete lack of the monocyte cluster (i.e. the upper white area pointed out by the arrow indicates the zone in which it should be visible);
(I) a medium-sized lymphocyte with abundant cytoplasm and circumferential projections (hairy cell); (L) another hairy cell, splenic marginal
zone lymphoma (SMZL): (M) WDF scattergram: an abnormal monocyte cluster (blue arrow) spreads over the pathological double-lymphocyte
cluster (indicated by the red arrow); (N) WNR scattergram: the monocyte cluster is clearly evident, as pointed out, with a regular pattern; (O)
WPC scattergram: same as above; (P) villous lymphocyte: it showed abundant cytoplasm, spongy chromatin and absent nucleoli; (Q) two
atypical lymphocytes with basophil cytoplasm and, one of them, with evident nucleolus.

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 Seghezzi et al.: WDF scattergram on suspect of hairy cell leukemia      3
May-Grünwald solution (Carlo Erba, Italy) and reviewed
by a laboratory specialist in hematology.
The OM review showed a complete lack of mono-
cytes and the presence of some hairy cells (3%) (Figure
1I, L). The clinical and laboratory data prompted clini-
cians to proceed with a bone marrow aspiration, which
showed (a) reduced representation of both erythroid,
granulocytic and megakaryocytes series, with mature
megakaryocytes and (b) diffused infiltrate of small and
medium-sized lymphoid elements (80%–90%) exhib-
iting a typical morphological pattern characteristic of
hairy cells. A further bone marrow biopsy showed (a)
bone marrow involvement in diffuse low-grade B-cell
lymphoma, with widespread infiltration of small-sized
cells with distinct nuclei and clear cytoplasm, whose
immunophenotyping was CD20 + /cyclin D1- (CCND1);
(b) increased representation of the megakaryocytic
series with mature and near-confluent megakaryocytes
with hyperlobulated nuclei; (c) increased representa-
tion of the erythroid series, with irregular erythroblas-
tic nests; (d) reduced representation of the granulocytic
series with reduced numbers of precursors; and (e) reti-
culin staining with a score of MF-2 [4].
The immunophenotyping on PB lymphocytes was
CD19 +, CD20 +, CD19/SIgλ +, CD19/CD103 +, CD19/
CD25 +, CD19/FMC7 +, CD19/CD11c +, CD3− and CD5−,
whereas the cytogenetic analysis of the bone marrow
and PB cells demonstrated the presence of BRAF V600E
mutation. Based on these results, the diagnosis of HCL
was made, according to the 2016 revision of the World
Health Organization classification of lymphoid neo-
plasms [4].
HCL is an uncommon (incidence: 2%–3%) hema-
tologic malignancy in the adult, characterized by pan-
cytopenia and marked susceptibility to infections [4,
5]. The patient usually presents signs and symptoms
related to the accumulation of abnormal B-lymphocytes
in the bone marrow. Abdominal lymphadenopathy is
common (10%), whereas skin and peripheral infiltration
(e.g. eye, kidney, pancreas, etc.) is relatively uncommon
(2%) [5, 6].
The diagnosis of HCL is fundamentally based on flow
cytometric immunophenotyping [4, 7].
The presence of hairy cells like (named villous cells)
on PB is not exclusive for HCL, but it could be observed
also in 50% of cases with the splenic marginal zone lym-
phoma (SMZL) [4, 8].
A typical case of SMZL in an adult patient is depicted
in Figure 1M–Q. The scattergrams and images were
taken from PB analysis on XN-module and OM smear
review of a patient with a B-cell SMZL confirmed by
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bone marrow biopsy and immunophenotyping (CD5−,
CD23−). Notably, the PB analysis on XN-module showed
a normal CBC, except for the thrombocytopenia, and the
alarm flag “Atypical Lympho?” (Table 1). The compari-
son of XN-module WDF, WNR and WPC scattergrams
(Figure 1M–O) in the SMZL patient versus a healthy adult
subject (Figure 1A–C) and the HCL patient (Figure 1F–H)
showed some important variations in the cell clusters
distribution. In the SMZL case, the WDF scattergram
(Figure 1M) displayed an abnormal lymphocyte double
cluster (colored in pink and indicated by the red arrow),
whereas the WNR and WPC scattergrams exhibited the
presence of the monocyte cluster (colored in green in
the WDF scattergram) (Figure  1N and O), as pointed
out by the black arrows. The PB smear review by OM
revealed an equal presence of atypical (3%) and villous
lymphocytes (3%), as in Figure 1P and Q. As for the HCL
case, it is noteworthy to mention that all the altera-
tions we previously evidenced in the WDF, WNR and
WPC scattergrams were shown in eight other HCL cases
that were diagnosed at our hospital facility during the
last year. These particular cell clustering patterns have
never been reported nor in normal subjects, neither in
other lymphoproliferative disorders (i.e. non-Hodgkin
lymphoma, multiple myeloma or acute leukemia) (data
not shown). About 50% of our cases did not show any
morphological flags (i.e. case described in this letter),
whereas the other part showed the flag “Blast/Abn_
Lympho?”. We hypothesized that the number of hairy
cells present in PB is crucial for triggering or not trig-
gering the flag. However, we have seen that there are
modifications of monocyte (MO) CPD provided by the
XN-module, like monocyte cells complexity (MO-X),
monocyte fluorescence intensity (MO-Y) and monocyte
cell size (MO-Z) closely related to the pathology. Specifi-
cally, we observed a significant decrease of the values
of these parameters compared with the reference range
(RR) reported in the literature [9]. The mean values are
as follows: MO-X = 109 (95% confidence interval [CI],
102–113) (RR, 114–122), MO-Y = 92.6 (95% CI, 79–100)
(RR, 102–127); and MO-Z = 58.3 (95% CI, 46–67) (RR, 59–
70). The MO CPD parameters could be a tool to improve
the quality of flagging for additional smear review.
In conclusion, data presented in this case-report
suggest the hypothesis that the presence of an abnormal
lymphocyte cell cluster distribution in WDF, WNR and
WPC scattergrams, and the modification of the CPD para-
meters, like those we could observe, is likely to be repre-
sentative of a pathological blood condition to be further
evaluated by the hemopathologist with an accurate OM
smear review, as a clue to the diagnosis of HCL.
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4      Seghezzi et al.: WDF scattergram on suspect of hairy cell leukemia
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization played no
role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
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