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

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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 bone marrow biopsy and immunophenotyping (CD5−,
by a laboratory specialist in hematology. CD23−). Notably, the PB analysis on XN-module showed
The OM review showed a complete lack of mono- a normal CBC, except for the thrombocytopenia, and the
cytes and the presence of some hairy cells (3%) (Figure alarm flag “Atypical Lympho?” (Table 1). The compari-
1I, L). The clinical and laboratory data prompted clini- son of XN-module WDF, WNR and WPC scattergrams
cians to proceed with a bone marrow aspiration, which (Figure 1M–O) in the SMZL patient versus a healthy adult
showed (a) reduced representation of both erythroid, subject (Figure 1A–C) and the HCL patient (Figure 1F–H)
granulocytic and megakaryocytes series, with mature showed some important variations in the cell clusters
megakaryocytes and (b) diffused infiltrate of small and distribution. In the SMZL case, the WDF scattergram
medium-sized lymphoid elements (80%–90%) exhib- (Figure 1M) displayed an abnormal lymphocyte double
iting a typical morphological pattern characteristic of cluster (colored in pink and indicated by the red arrow),
hairy cells. A further bone marrow biopsy showed (a) whereas the WNR and WPC scattergrams exhibited the
bone marrow involvement in diffuse low-grade B-cell presence of the monocyte cluster (colored in green in
lymphoma, with widespread infiltration of small-sized the WDF scattergram) (Figure  1N and O), as pointed
cells with distinct nuclei and clear cytoplasm, whose out by the black arrows. The PB smear review by OM
immunophenotyping was CD20 + /cyclin D1- (CCND1); revealed an equal presence of atypical (3%) and villous
(b) increased representation of the megakaryocytic lymphocytes (3%), as in Figure 1P and Q. As for the HCL
series with mature and near-confluent megakaryocytes case, it is noteworthy to mention that all the altera-
with hyperlobulated nuclei; (c) increased representa- tions we previously evidenced in the WDF, WNR and
tion of the erythroid series, with irregular erythroblas- WPC scattergrams were shown in eight other HCL cases
tic nests; (d) reduced representation of the granulocytic that were diagnosed at our hospital facility during the
series with reduced numbers of precursors; and (e) reti- last year. These particular cell clustering patterns have
culin staining with a score of MF-2 [4]. never been reported nor in normal subjects, neither in
The immunophenotyping on PB lymphocytes was other lymphoproliferative disorders (i.e. non-Hodgkin
CD19 +, CD20 +, CD19/SIgλ +, CD19/CD103 +, CD19/ lymphoma, multiple myeloma or acute leukemia) (data
CD25 +, CD19/FMC7 +, CD19/CD11c +, CD3− and CD5−, not shown). About 50% of our cases did not show any
whereas the cytogenetic analysis of the bone marrow morphological flags (i.e. case described in this letter),
and PB cells demonstrated the presence of BRAF V600E whereas the other part showed the flag “Blast/Abn_
mutation. Based on these results, the diagnosis of HCL Lympho?”. We hypothesized that the number of hairy
was made, according to the 2016 revision of the World cells present in PB is crucial for triggering or not trig-
Health Organization classification of lymphoid neo- gering the flag. However, we have seen that there are
plasms [4]. modifications of monocyte (MO) CPD provided by the
HCL is an uncommon (incidence: 2%–3%) hema- XN-module, like monocyte cells complexity (MO-X),
tologic malignancy in the adult, characterized by pan- monocyte fluorescence intensity (MO-Y) and monocyte
cytopenia and marked susceptibility to infections [4, cell size (MO-Z) closely related to the pathology. Specifi-
5]. The patient usually presents signs and symptoms cally, we observed a significant decrease of the values
related to the accumulation of abnormal B-lymphocytes of these parameters compared with the reference range
in the bone marrow. Abdominal lymphadenopathy is (RR) reported in the literature [9]. The mean values are
common (10%), whereas skin and peripheral infiltration as follows: MO-X = 109 (95% confidence interval [CI],
(e.g. eye, kidney, pancreas, etc.) is relatively uncommon 102–113) (RR, 114–122), MO-Y = 92.6 (95% CI, 79–100)
(2%) [5, 6]. (RR, 102–127); and MO-Z = 58.3 (95% CI, 46–67) (RR, 59–
The diagnosis of HCL is fundamentally based on flow 70). The MO CPD parameters could be a tool to improve
cytometric immunophenotyping [4, 7]. the quality of flagging for additional smear review.
The presence of hairy cells like (named villous cells) In conclusion, data presented in this case-report
on PB is not exclusive for HCL, but it could be observed suggest the hypothesis that the presence of an abnormal
also in 50% of cases with the splenic marginal zone lym- lymphocyte cell cluster distribution in WDF, WNR and
phoma (SMZL) [4, 8]. WPC scattergrams, and the modification of the CPD para-
A typical case of SMZL in an adult patient is depicted meters, like those we could observe, is likely to be repre-
in Figure 1M–Q. The scattergrams and images were sentative of a pathological blood condition to be further
taken from PB analysis on XN-module and OM smear evaluated by the hemopathologist with an accurate OM
review of a patient with a B-cell SMZL confirmed by smear review, as a clue to the diagnosis of HCL.

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Lab Hematol 2016;38:160–6.
manuscript and approved submission.
4. Foucar K, Falini B, Catovsky D, Stein H. Hairy cell leukemia. In:
Research funding: None declared. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H,
Employment or leadership: None declared. et al., editors. WHO classification of tumours of haematopoietic
Honorarium: None declared. and lymphoid tissues, 4th ed. France: International Agency for
Competing interests: The funding organization played no Research on Cancer, 2008:188–90.
5. Grever MR, Abdel-Wahab O, Andritsos LA, Banerji V, Barrientos
role in the study design; in the collection, analysis, and
J, Blachly JS, et al. Consensus guidelines for the diagnosis and
interpretation of data; in the writing of the report; or in the
management of patients with classic hairy cell leukemia. Blood
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