Extreme macrocytosis in a Shiba dog
S. Neo1, E. Ogawa2, S. Yamashiro1, D. Suzuki1, Taro Ayabe3, M. Hisasue1, R. Tsuchiya1
Laboratory of Internal Medicine 21 and Basic Education2, Azabu University, Kanagawa, Japan
Ozenji Veterinary Hospital3
Abstract
Results
Table 1. Patient’s laboratory data
※Biochemistry exhibit no abnormalities.
Erythrocytes in healthy Shiba dogs (Shibas) are typically microcytic in relation to
other dog breeds. Approximately 10% of them have erythrocytes with high K, low Na,
Hematology
Patient
Reference interval
Urinalysis
and high glutathione (GSH) concentrations, called HK/HG cells. HK/HG cells tend to
8.58
6.0-17.0
Color
Yellow
Urine sediment
WBC (×103/uL)
have increased osmotic fragility and higher MCV (76.0±5.1 fl (mean±SD)) than
HK/LG cells (high K, low Na, and low GSH) at 65.0-68.0 fl, or LK cells(low K, high Na,
169
200-500
Transparency
Clear
WBC
3+
Platelet (×103/uL)
and low GSH) at 65.7±4.1 fl.
5.32
5.5-5.8
Sp.Gr.
1.041
RBC
RBC (×106/uL)
Aquaporin1, the major water channel in RBCs, has been thought to function primarily
HCT (%)
50.9
37-55
Protein
1+
Crystals
1+ (Bilirubin)
to facilitate the transmembrane transport of water in response to osmotic gradients.
Hgb (g/dL)
12.3
12-18
Bilirubin
3+
We report an extreme macrocytosis compared to those of typical Shiba HK/HG cells,
found in a one year old Shiba with a history of hemoglobinuria of sudden onset.
5.3
Blood
Reticulocytes (×104/uL)
Hematological abnormalities included decreased RBC count (5.32; reference interval
MCV (fL)
95.7
66-77
Patient
Reference intervals
(RI) 5.5-8.5×106/uL ), increased MCV (95.7; RI 66-77fL), decreased MCHC (24.2: RI
3
32-36g/dL), and decreased platelet count (169; RI 200-500×10 /uL ). Reticulocyte
MCH (pg)
23.1
19.9-24.5
Cobalamin (pg/mL)
1050
233-914
count was not increased (53200/uL ). Serum cobalamin was mildly elevated.
MCHC (g/dL)
24.2
32-36
Folate (ng/mL)
14.0
3.6-12.9
Since the macrocytosis was not attributable to reticulocytosis or cobalamin deficiency,
RBC morphology
Ghost cells 2+
other potential causes of macrocytosis were investigated. The osmotic fragility was
markedly increased (Parpart method). The erythrocytes exhibited high K (114
Macrocytosis without increased polychromatophils was found. Few ghost cells were seen on the blood smear, which could
[7.4±0.9mmol/L]), low Na(<10 [116.1±6.6mmol/L]), and high GSH (50
suggest hemolysis. Although occult blood test was negative with dipstick test, marked bilirubinuria was detected. Cobalamin
[7.0±0.3umol/gHb]) concentrations within erythrocytes, indicating HK/HG cells.
Erythrocyte membrane transporter Aquaporin1 (AQP1) was analyzed by
and Folate concentrations were not decreased.
immunoblotting and expressed approximately 2 times higher levels compared to HK
cells from other Shibas.
Table 2. Concentrations of K+, Na+,
Table 3. Activities of glutathione
Fig 1. Blood smear
Findings suggest that in addition to the characteristics of HK/HG, increased
and GSH in the patient’s erythrocytes
reductase (GR) and pyruvate kinase (PK)
levels of AQP1 may have contributed to macrocyte formation in this dog possibly
in the patient’s erythrocytes
Patient
Mean ±SD
through increased water flow into the erythrocytes.
Introduction
Our patient, one year old male Shiba presented with a history of
hemoglobinuria of sudden onset showed …
a significant macrocytosis (MCV: 95.7): much larger than that of
HK/HG cells.
Few ghost cells and bilirubinuria, which suggests hemolysis.
Ghost cells (arrow) are found.
K+ (mmol/L)
114
7.4±0.9
Na+(mmol/L)
<10
116.1±6.6
GSH umol/gHb
50
7.0±0.3
Patient’s erythrocytes contained high K+,
Low Na+, and high GSH. ⇒HK/HG cells
Fig 2. Osmotic fragility
Objective
To investigate the potential cause to induce extreme
macrocytosis with possible hemolysis in a Shiba.
Questions:
Are they HK/HG cells?
Are they immature erythrocytes?
Is the AQP-1 expression associated with their cellular volume?
GR
PK
K+
Na+
GSH
AQP-1
① Laboratory data: CBC, Urinalysis,
Cobalamin, Folate
② Osmotic fragility: Parpart method
③ Concentrations within erythcotyes: K+,
Na+, GSH, GR, PK
④ AQP-1: Immunoblotting
31.3kDa
25.4kDa
Osmotic fragility of the patient showed
marked increased compared to that of
healthy beagle and HK/HG dogs.
Mean ±SD
GR IU/gHb
2.45
3.52±0.73
PK IU/gHb
10.4
8.3±2.2
GR and PK activities were within reference
interval
⇒Patient’s erythrocytes were not immature.
Fig 3. Immunoblotting of erythrocyte
membrane isolated from patient and
healthy HK/HG Shibas
47.3kDa
Materials & Methods
Patient
glycosilated
AQP1
unglycosilated
AQP1
AQP-1 of the patient expressed
approximately 2 times higher than that
of HK/HG shiba dogs.
Summary
Erythrocytes
of the patient Shiba
References
were HK/HG cells.
Erythrocytes of the patient Shiba was
not immature erythrocytes.
Increased levels of AQP-1 may
contribute to macrocyte formations
through increased water permeability.
↑AQP-1
Water?
Water?
Macrocyte
Hemolysis
Water?