SPURIOUS WHITE BLOOD CELLS SCATTERGRAM AND DISCREPANCY IN WHITE
BLOOD CELLS COUNTS IN ONE DOG
Laetitia Piane1, Rachel Lavoué2, Cathy Trumel1
1 : Equipe de Biologie médicale-Histologie, CREFRE, Université de Toulouse, INSERM,
UPS, ENVT, Toulouse, France
2 : Unité de Médecine Interne, Université de Toulouse, UPS, INP, ENVT, F-31076 Toulouse,
France
Signalement
A 7-years old neutered male Labrador retriever dog.
Clinical History
The dog was referred to the internal medicine unit for exploration of abdominal petechia,
edemas and acute hematochezia.
Clinical findings
Physical examination revealed hyperthermia (39.6°C), tachypnea, tachycardia and congested
mucous membranes. Caudal abdominal palpation was painful and an oblong mass was
identified. On numerous occasions edema and abdominal and thoracic effusions were
observed. The dog vomited twice during the consultation. Hematochezia was objectified by
rectal palpation. Buccal examination revealed slight gingival bleeding.
Diagnostic procedures
Biochemistry, coagulation and hematology panels were performed. Results are displayed in
tables 1, 2 and 3. Plasma biochemistry and coagulation profiles were unremarkable. Complete
blood cell count (CBC) revealed a moderate normocytic normochromic, non regenerative
anemia, a leukocytosis with marked discrepancy between the white blood cell (WBC) counts
given by the BASO channel (32.1 109/L) and the DIFF channel (66.5 109/L) and an abnormal
WBC-DIFF scattergram. On the left part of the WBC-DIFF channel scattergram, there was a
continuum between the debris area and the neutrophils cluster with an unclear separation
between neutrophils, lymphocytes and monocytes clusters and a grey zone including the
debris area, the neutrophils cluster and a part of the lymphocyte cluster (Figure 1). However,
the BASO channel scattergram was normal. Blood smears revealed numerous neutrophils
with band cells and some circulating mast cells. Red blood cells (RBCs) were moth-eaten and
modified in shape, with an amorphous aggregated pinkish to basophilic material in the
background distorting RBCs contours especially at the edges of the blood smear (Figure 2),
which is highly suggestive of cryoglobulins.
Treatment and following:
The dog was hospitalized and received a Ringer Lactate perfusion with potassium
supplementation. Glucocorticoids (prednisolone) were administered intravenously at a dose of
1 mg/kg.
To explore the hypothesis of cryoglobulins, three new blood specimens were taken for
hematology and CBC and blood smears were performed after putting the specimens at 4°C,
room temperature, and 37°C for 30 minutes. Results are displayed in Table 4. At 4°C and
room temperature, a slight discrepancy between leukocyte count given by the BASO channel
and the DIFF channel was observed as well as an abnormal scattergram characterized by
numerous pink dots in the upper part of the DIFF and a continuum between the debris area
and the neutrophils, the lymphocytes and the monocytes cluster. The CBCs at 4°C and room
temperature also revealed a monocytosis. The same abnormalities as previously described
were observed on blood smears. At 37°C, the leukocyte count given by the BASO channel
and the DIFF channel were almost similar. On the WBC-DIFF scattergram, pink dots at the
top of the graph were less numerous and suggestive of reactive lymphocytes. The continuum
between the debris area and the neutrophils cluster was not observed. An unclear separation
between neutrophils and lymphocytes clusters is still observed, suggestive of the presence of
band cells or toxic neutrophils. Blood smear revealed normally shaped RBCs without
amorphous basophilic material, which confirmed the suspicion of cryoglobulins (Figure 4).
Because of deterioration of body condition, the dog was euthanized. Autopsy and histology
were performed and revealed a septic neutrophilic, necrotizing and thrombotic systemic
vasculitis associated with severe, diffuse, subacute, fibrinous and septic pericarditis.
Interpretation:
Abnormal WBC-DIFF scattergram and marked discrepancy between the WBC counts given
by the DIFF channel and the BASO channel secondary to cryoglobulins.
Discussion & conclusion
The case describes a dog with cryoglobulinemia, erroneously high WBC count given by the
DIFF channel and abnormal WBC-DIFF scattergram. Cryoglobulinemia was confirmed by
erroneous results and the detection of precipitates on blood smears at 4°C and room
temperature which were not apparent when blood had been heated to 37°C
There are only few reports on cold agglutinins in dogs and, to our knowledge; this is the first
case of cryoglobulinemia [1, 2]. Cryoglobulins are serum immunoglobulins or
immunoglobulin complexes that undergo reversible precipitation at low temperatures.
According to classification of Brouet et al. (1974), three types of cryoglobulins are described
[3, 4]:
- Type 1 (10-15% of the total cases): A single monoclonal immunoglobulin, characteristically
found in patients with multiple myeloma or Waldenstrom's macroglobulinaemia; usually IgM,
less frequently IgG, and rarely IgA.
- Type 2 (50-60% of the total cases): One monoclonal and one polyclonal fraction (mixed
cryoglobulins); IgM-IgG is the combination usually found.
- Type 3 (25-30% of the total cases): Mixed polyclonal cryoglobulins, associated with
infection or inflammatory disease or, less commonly, idiopathic.
In our case, cryoglobulinemia was probably Type 3 cryoglobulinemia secondary to severe,
systemic and septic inflammatory process.
In human medicine, erroneously high WBC counts were reported to be due to platelet
aggregates and large platelets, nucleated red blood cells, red blood cells resistant to lysis,
cryoglobulins, cryofibrinogen, lipids, microorganisms, adipose tissue or overfilling blood
collection vacuum tubes [5, 6, 7]. The presence of cryoglobulins was first reported as causing
erroneous WBC counts, but it also leads in many instances to spuriously elevated PLT counts,
and at times to decreased RBC counts or Hb measurements. Abnormalities generated must be
considered because they have particular characteristics, and their recognition may be the first
clue leading to the diagnosis of cryoglobulinemia [5, 7, 8, 9, 10, 11].
The degree of interference in WBC or platelet counts seems to depend not only on the
instrument used but also on the type of precipitate. Indeed, spuriously elevated cell counts are
due to the presence of particles of cryoglobulins being counted as WBCs or platelets in
relation to such physical properties as their size, structure, and shape.
In human medicine, hematology analyzers based on an impedance method, could detect
cryoglobulins as small particles (<35 fL) at the left of the lymphocyte monocyte granulocyte
diagram, leading to spuriously elevated WBC counts. The presence of particles smaller than
35 fL due to cryoglobulins producing a characteristic 'high take-off' on the WBC histogram
with Coulter counter [4]. With hematology analyzers based on an optical method,
cryoglobulins could also appear on the DIFF channel scattergram, leading to falsely elevated
WBC counts. A cloud of small dots may be observed on the WBC DIFF channel scattergram,
near the lymphocytes or above the threshold for platelet and debris, or at the top of the
neutrophils cloud (ADVIA, Technicon). The BASO channel, analyzing WBCs according to
their volume following differential cytoplasmic stripping, is not affected by cryoglobulins.
The smaller cryoglobulins interfere on the histogram of platelet volume distribution,
generating a characteristic triangular figure and an alarm is usually generated [11]. Platelet
counts are spuriously elevated, and the mean platelet volume is unrealistically low (too small
for true platelets) [11]. In our case, cryoglobulins were observed on the DIFF channel
scattergam as a grey zone with a continuum including the debris area, the neutrophils cluster
and a part of the lymphocyte cluster or by numerous pink dots in the upper part of the graph
and a continuum between the debris area, the neutrophils, the lymphocytes and the monocytes
clusters on the left part of the scattergram.
In human medicine, cryoprecipitates may be observed in some instances on stained blood
smears. Various morphological aspects were reported, including dense amorphous clusters or
flake-like particles, needle-shaped crystals, pinkish globules or invisible precipitates that
change the morphology of RBCs into a ‘moth-eaten’ aspect [9, 11, 12, 13, 14]. One study on
4 cases of cryoglobulinemia in human patients revealed that the morphological aspect is
correlated with hematologic abnormalities. Indeed, the cryoglobulin particles or clusters
which appear like pinkish amorphous deposits between RBCs on blood smears or fusiform to
needle-shape crystals on contrast microscope interfered in WBC counts and the cryoglobulin
particles which appear like thin refringent precipitates or flakes of clear amorphous material
on contrast microscope interfered in platelet counts [11]. In our case, cryoglobulins were
identified as dense amorphous pinkish to basophil clusters on the blood smears performed
during the follow-up, which could explain why WBC count was affected but not platelet
count.
As cryoglobulins are immunoglobulins that precipitate at temperature lower than 37°C,
abnormal counts are observed mainly on hematology analyzers that both use reagents and
perform analysis at room temperature. However, hematology analyzers that use prewarmed
reagents, and a WBC diluent at low pH reagents (for example ADVIA 120, Bayer) that
normally avoid precipitation of cryoglobulins, are not free of abnormalities and spurious
counts are also observed [5, 10, 11]. When specimens are warmed to 37°C for at least 30 min
and reanalyzed promptly abnormalities disappear. If this is not sufficient (partial redissolution
occurs) a new specimen must be taken in warmed tubes maintained at 37°C until analysis to
obtain accurate CBC counts [5, 10, 11, 12, 15].
Cryoglobulins could also modified hemoglobin and RBC counts related to disturbance of light
transmittance or a flow anomaly. Cryoglobulins may form a gel leading to high viscosity of
the specimen and aspiration of an unrepresentative aliquote. However, spurious hemoglobin
measurement is far from being a consistent finding in the presence of cryoglobulins [11].
Laboratory recognition of the cryoglobulins is important to correct factitious results with
automated blood cell counters, mainly pseudoleukocytosis and pseudothrombocytosis.
Moreover cryoglobulin-induced laboratory artifacts may be the first factor prompting the
assessment for cryoglobulinemia and the diagnosis of the underlying cause. Of clinical
interest, cryoglobulinemia may be diagnosed several years before the underlying cause,
especially for hematologic diseases [11].
Table 1: Complete blood cell count results
Analyte
Results
Reference intervals (Sysmex)
WBC (109/L)
32.1
5.6-20.4
D-WBC (109/L)
66.5
ND*
NEUT (109/L)
/
2.9-13.6
LYMPH (109/L)
/
1.1-5.3
MONO (109/L)
/
0.4-1.6
EOS (109/L)
/
0.1-1.5
RBC (1012/L)
5
5.2-7.9
HGB (g/dL)
12.1
12.4-19.2
HCT (L/L)
32.2
35-52
MCV (fL)
64.7
60-71
MCHC (g/dL)
37.6
34.4-38.1
PLT-I (109/L)
371
64-613
PLT-O (109/L)
352
108-562
RET (109/L)
28.9
19.4-150.1
* ND not determined
Table 2: Plasma biochemistry results
Analyte
Results
Reference intervals (Vitros)
Creatinine (µmol/L)
116
44-133
Total proteins (g/L)
66
48-66
Albumin (g/L)
33.3
23-39
ALP (U/L)
43
20-155
ALT (U/L)
26
3-50
Sodium (mmol/L)
144
138-148
Potassium (mmol/L)
4.6
3.2-5.0
Chloride (mmol/L)
115
110-118
Bicarbonates (mmol/L)
18
16-25
Glucose (g/L)
1.42
0.7-1.8
Table 3: Coagulation profile results
Analyte
Results
Reference intervals (Stago)
Fibrinogen (g/L)
2.3
1.3-4.8
PT (s)
8.4
7.1-9
aPTT (s)
11.3
12.8-17.2
A
B Figure 1: Comparison of the WBC scattergrams by the DIFF (A) and BASO (B) channels in
a case of canine cryoglobulinemia.
On the left part of the WBC-DIFF channel scattergram (A), we observed a grey zone with a continuum
including the debris area, the neutrophils cluster and a part of the lymphocyte cluster (red arrow).
There is also an unclear separation between neutrophils, lymphocytes and monocytes clusters (red
circle).
The BASO channel scattergram was not altered (B).
Figure 2: Blood smear from the dog. x40 Objective. May Grünwald Giemsa.
We observed an amorphous basophilic material in the background (black arrows) which modifies the
RBCs shape (red arrow).
Table 4: Comparison of hematology results at 4°C, room temperature and 37°C
Analyte
4°C
room
37°C
temperature
Reference intervals at
room temperature
WBC (109/L)
31.1
30.6
31.9
5.6-20.4
D-WBC (109/L)
34.8
36.1
30.7
ND*
NEUT (109/L)
24.6
24.2
29.4
2.9-13.6
LYMPH (109/L)
4.0
3.0
0.8
1.1-5.3
MONO (109/L)
2.5
3.3
1.6
0.4-1.6
EOS (109/L)
0.0
0.1
0.1
0.1-1.5
RBC (1012/L)
3.6
3.6
3.5
5.2-7.9
HGB (g/dL)
8.6
8.5
8.5
12.4-19.2
HCT (L/L)
24.1
24.1
23.7
35-52
MCV (fL)
67.7
67.9
67.5
60-71
MCHC (g/dL)
35.7
35.3
35.9
34.4-38.1
387
394
394
64-613
406
429
415
108-562
84.7
69.9
69.1
19.4-150.1
9
PLT-I (10 /L)
9
PLT-O (10 /L)
9
RET (10 /L)
* ND not determined
A B
C Figure 3: WBC scattergram from the dog at 4°C (A), room temperature (B) and 37°C
(C)
At 4°C and room temperature, we observed an abnormal WBC-DIFF scattergram characterized by
numerous pink dots in the upper part of the DIFF and a continuum between the debris area and the
neutrophils, the lymphocytes and the monocytes cluster (red arrows). After heating at 37°C, the
continuum between debris areas and other clusters was not observed (black arrow). There are few pink
dots at the top of the graph, which could be suggestive of reactive lymphocytes, and unclear separation
between neutrophils, lymphocytes and monocytes clusters persists, which could be suggestive of band
cells or toxic neutrophils (red circle).
Figure 4: Blood smear from the dog at 38°C. x40 Objective. May Grünwald Giemsa.
RBCs abnormalities and amorphous basophilic materiel in the background observed at room
temperature (Figure 2) disappear.
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