Undergraduate Course in Veterinary Clinical Pathology
Socrates-Erasmus Programme
Haematology
LECTURE 5.
5 EVALUATION OF BLOOD
SMEAR RBCs AND RETICULOCYTES
5-1
OVERVIEW
1. RBC morphological evaluation
2. Reticulocyte count and evaluation
5-2
1. RBC MORPHOLOGICAL
EVALUATION
5-3
VARIATIONS IN SPECIES
CAT
DOG
-Large erythrocytes
-Size uniformity
-Central pallor
-Smaller erythrocytes
-Anisocytosis
-Scarce central pallor
5-4
VARIATIONS IN SPECIES
HORSES
RUMINANTS
Rouleaux
(sedimentation tendency)
Anisocytosis and crenation
5-5
EVALUATION OF RBC NUMBER AND SIZE IN
DIFFERENT ANIMAL SPECIES
100
30
90
25
80
70
60
MCV fl
12
RBC 10 /l
20
15
50
40
10
30
20
5
10
0
n=
46
61
Hund
dogs
Katze
cats
105
176
Pferd Schwein
horses
pigs
Tierart
species
141
Rind
cattle
NUMBER
181
Schaf
sheep
172
Ziege
goats
0
n=
46
Hund
dogs
61
105
Katze horses
Pferd
cats
176
141
181
172
Schwein
Rind sheep
Schaf goats
Ziege
pigs cattle
Tierart
species
VOLUME
5-6
ABNORMALITIES OF RBCs MORPHOLOGY
1. Abnormalities in size
-MACROCYTOSIS
-MICROCYTOSIS
2. Abnormalities in colour
-POLYCHROMASIA
-HYPOCHROMASIA
3. Abnormalities in shape
-SPHEROCYTOSIS
-NON-SPECIFIC FINDINGS
4. Abnormalities in distribution
-ROULEAUX
-AGGLUTINATION
5. Inclusion Bodies and infectious agents
5-7
ABNORMALITIES IN SIZE
MACROCYTOSIS
MICROCYTOSIS
Reticulocytes
FeLV
Iron deficiency
5-8
ABNORMALITIES IN SIZE
Reticulocytes and spherocytes
in a dog with immune-mediated
haemolytic anemia
Macrocytic (but non-regenerative)
anemia in FeLV infected cat
5-9
ABNORMALITIES IN COLOUR
POLYCHROMASIA
HYPOCHROMASIA
Reticulocyte
Iron deficiency
5-10
ABNORMALITIES IN SHAPE
(POIKILOCYTOSIS)
-SPHEROCYTOSIS (MARKER OF IMMUNE-MEDIATED HAEMOLYTIC
ANAEMIA)
-NON-SPECIFIC FINDINGS
- Crenation
- Acanthocyte
- Schistocyte/fragmentocyte
- Target Cell
5-11
Spherocytes
5-12
Crenation
Numerous pin-point projections
5-13
Acanthocytes (few irregular elongations of RBC border with
rounded ends often forming a bud)
5-14
Schistocytes. (erythrocyte fragmentation)
5-15
Target cell
5-16
ABNORMALITIES IN RBC DISTRIBUTION
Rouleaux
- normal finding in horses
- indicates inflammation
in small animals
5-17
Agglutination
Macroscopic
Microscopic
- can suggest immune-mediated haemolytic anaemia
5-18
INCLUSION BODIES AND INFECTIOUS
AGENTS
HOWELL-JOLLY BODIES
BASOPHILIC STIPPLING
Signs of
regeneration
NUCLEATED ERYTHROCYTES
HEINZ BODIES
INFECTIOUS AGENTS (Babesia, Mycoplasma haemofelis)
5-19
Nucleated RBCs
and Howell-Jolly
Bodies
5-20
Basophilic stippling
5-21
Heinz
Bodies
5-22
Heinz
Bodies
5-23
Infectious agents
Babesia canis
Haemobartonella felis
(Mycoplasma haemofelis)
5-24
2. RETICULOCYTE COUNT AND
EVALUATION
5-25
Reticulocytes are young (immature) erythrocytes prematurely
released to blood from the bone marrow in some anaemias.
CLINICAL APPLICATIONS:
Evaluation of erythropoiesis in bone narrow.
Differentiation of regenerative and non
non-regenerative
regenerative anaemia
anaemia.
TECHNIQUES OF DETECTION:
-Romanowsky stains
-Supravital stains:
• New methylene blue
• Brillant cresyl blue
5-26
ROMANOWSKY STAINS
In dogs, an average of >10 polychromatic red cells per OIF
suggests a marked regenerative response
5-27
NEW METHYLENE BLUE (NMB) STAIN
Reticulocytes are non-nucleated erythrocytes in which NMB
stain precipitates in RNA-protein complexes
5-28
ABSOLUTE RETICULOCYTE
COUNT
- Manual
- Automated
A t
t d (some
(
haematological
h
t l i l analysers)
l
)
5-29
MANUAL ABSOLUTE RETICULOCYTE
COUNT
1. Count number of reticulocytes per 500-1000 erythrocytes
(blood smear)
-e.g. 10 reticulocytes / 500 erythrocytes
2 Calculate % of reticulocytes
2.
-e.g. Reticulocyte % = 10 x 100 / 500 = 2
3. Calculate absolute reticulocyte count based on
erythrocyte count from the haematology analyser
-e.g. Absolute reticulocyte count (109/L)= Reticulocyte %
x Erythrocyte count / 100
5-30
INTERPRETATION
If absolute reticulocyte count:
Dogs >60x 109/L
A sign of regeneration
Cats>50x 109/L
5-31
RETICULOCYTE PRODUCTION INDEX:
CALCULATIONS
- Corrected reticulocyte percentage (CRP)
CRP= % reticulocytes x PCV of sample/normal PCV
- Reticulocyte Production Index (RPI)
RPI= CRP/Maturation Index(MI)
(values for dogs)
PCV
0.45
0.35
0.25
0.15
MI (days)
1
1.5
2
2.5
5-32
RETICULOCYTE PRODUCTION INDEX:
PRACTICAL EXAMPLE
Dog with PCV=0.15L/L and reticulocytes =15%
- Corrected reticulocytes percentage (CRP)
CRP= 0.15 x 0.15/0.45 = 5%
- Reticulocyte Production Index (RPI)
RPI= 5/2.5 =2
5-33
INTERPRETATION
RPI > 3
Very good regeneration
RPI = 1-3
RPI < 1
Good regeneration
Inadequate regeneration
5-34
EXAMPLES OF USE OF RETICULOCYTE
PARAMETERS IN DOGS
Units
Case 1
Case 2
Case 3
L/L
0.15
0.15
0.15
RBC count
x1012/L
2.0
2.0
2.0
Reticulocytes
y
%
3
12
25
60
240
500
Hct/PCV
Reticulocytes x109/L
CRP
---
1
4
8.33
RPI
---
0.4
1.6
3.33
Diagnosis
nongood
highly
regenerative regenerative regenerative
anemia
anemia
anemia
5-35
SPECIES VARIATIONS: IN CLINICALLY
HEALTHY ANIMALS
- Dogs. Low number of reticulocytes (<1%), aggregate only
- Cats.
Cats Two types of reticulocytes:
• aggregate: blue stained coarse clumping (0.5% of erythrocytes)
• punctate: small, blue stained dots (1-10%).
- Ruminants and horses. Virtually no reticulocytes in blood.
5-36
SPECIES VARIATION: in anaemic
animals
- Canine. Strong reticulocyte response in regenerative
anaemias.
Aggregated reticulocytes (indicate recent response)
- Feline.
Punctated reticulocytes (indicate response to anaemia
occurring 3-4 weeks previously)
- Ruminants and horses. Reticulocytes may not appear
even in very severe anaemias
5-37