LECTURE 5 EVALUATION OF BLOOD

Undergraduate Course in Veterinary Clinical PathologySocrates-Erasmus Programme

Haematology

5-1

LECTURE 5. EVALUATION OF BLOOD SMEAR RBCs AND RETICULOCYTES

OVERVIEW

1. RBC morphological evaluation

5-2

2. Reticulocyte count and evaluation

1. RBC MORPHOLOGICAL EVALUATION

5-3

EVALUATION

DOG CAT

VARIATIONS IN SPECIES

5-4

-Large erythrocytes-Size uniformity-Central pallor

-Smaller erythrocytes-Anisocytosis

-Scarce central pallor

HORSES RUMINANTS

VARIATIONS IN SPECIES

5-5

Rouleaux(sedimentation tendency) Anisocytosis and crenation

EVALUATION OF RBC NUMBER AND SIZE INDIFFERENT ANIMAL SPECIES

20

25

30

70

80

90

100

5-6

NUMBER VOLUME

0

5

10

15

RBC

1012

/l

Hund Katze Pferd Schwein Rind Schaf ZiegeTierart

n = 46 6161 105 176 141 181 1720

10

20

30

40

50

60

MC

V fl

Hund Katze Pferd Schwein Rind Schaf ZiegeTierart

n = 46 6161 105 176 141 181 172

dogs cats horses pigs cattle sheep goatsspecies

dogs cats horses pigs cattle sheep goatsspecies

ABNORMALITIES OF RBCs MORPHOLOGY1. Abnormalities in size

-MACROCYTOSIS-MICROCYTOSIS

2. Abnormalities in colour -POLYCHROMASIA-HYPOCHROMASIA

5-7

-HYPOCHROMASIA 3. Abnormalities in shape

-SPHEROCYTOSIS -NON-SPECIFIC FINDINGS

4. Abnormalities in distribution -ROULEAUX-AGGLUTINATION

5. Inclusion Bodies and infectious agents

ReticulocytesMACROCYTOSIS

FeLV

ABNORMALITIES IN SIZE

5-8

MICROCYTOSIS Iron deficiency

ABNORMALITIES IN SIZE

5-9

Reticulocytes and spherocytesin a dog with immune-mediatedhaemolytic anemia

Macrocytic (but non-regenerative)anemia in FeLV infected cat

ABNORMALITIES IN COLOUR

POLYCHROMASIA HYPOCHROMASIA

5-10Reticulocyte Iron deficiency

-SPHEROCYTOSIS (MARKER OF IMMUNE-MEDIATED HAEMOLYTICANAEMIA)

-NON-SPECIFIC FINDINGS

ABNORMALITIES IN SHAPE (POIKILOCYTOSIS)

5-11

- Crenation- Acanthocyte- Schistocyte/fragmentocyte- Target Cell

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

Rouleaux

- normal finding in horses- indicates inflammationin small animals

ABNORMALITIES IN RBC DISTRIBUTION

5-17

in small animals

Agglutination

Macroscopic Microscopic

5-18

- can suggest immune-mediated haemolytic anaemia

HOWELL-JOLLY BODIES

BASOPHILIC STIPPLING

NUCLEATED ERYTHROCYTES

INCLUSION BODIES AND INFECTIOUS AGENTS

Signs ofregeneration

5-19

HEINZ BODIES

INFECTIOUS AGENTS (Babesia, Mycoplasma haemofelis)

Nucleated RBCs

5-20

and Howell-JollyBodies

Basophilic stippling

5-21

HeinzBodies

5-22

HeinzBodies

5-23

Infectious agents

5-24

Babesia canis Haemobartonella felis(Mycoplasma haemofelis)

2. RETICULOCYTE COUNT AND EVALUATION

5-25

EVALUATION

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 regenerative anaemia

5-26

TECHNIQUES OF DETECTION: -Romanowsky stains-Supravital stains:

• New methylene blue • Brillant cresyl blue

Differentiation of regenerative and non-regenerative anaemia.

ROMANOWSKY STAINS

5-27

In dogs, an average of >10 polychromatic red cells per OIF suggests a marked regenerative response

NEW METHYLENE BLUE (NMB) STAIN

Reticulocytes are non-nucleated erythrocytes in which NMBstain precipitates in RNA-protein complexes

5-28

ABSOLUTE RETICULOCYTE COUNT

- Manual

A t t d ( h t l i l l )

5-29

- Automated (some haematological analysers)

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

5-30

2. Calculate % of reticulocytes

-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

If absolute reticulocyte count:

INTERPRETATION

5-31

Dogs >60x 109/LA sign of regeneration

Cats>50x 109/L

RETICULOCYTE PRODUCTION INDEX:CALCULATIONS

- Corrected reticulocyte percentage (CRP)

CRP= % reticulocytes x PCV of sample/normal PCV

5-32

- Reticulocyte Production Index (RPI)

RPI= CRP/Maturation Index(MI) PCV MI (days)0.45 1

(values for dogs) 0.35 1.50.25 20.15 2.5

- Corrected reticulocytes percentage (CRP)

Dog with PCV=0.15L/L and reticulocytes =15%

RETICULOCYTE PRODUCTION INDEX:PRACTICAL EXAMPLE

5-33

CRP= 0.15 x 0.15/0.45 = 5%

- Reticulocyte Production Index (RPI)

RPI= 5/2.5 =2

INTERPRETATION

RPI > 3 Very good regeneration

RPI = 1-3 Good regeneration

5-34

RPI = 1-3 Good regeneration

RPI < 1 Inadequate regeneration

EXAMPLES OF USE OF RETICULOCYTE PARAMETERS IN DOGS

Units Case 1 Case 2 Case 3

Hct/PCV L/L 0.15 0.15 0.15 RBC count x1012/L 2.0 2.0 2.0 Reticulocytes % 3 12 25

5-35

y % 3 12 25 Reticulocytes x109/L 60 240 500 CRP --- 1 4 8.33 RPI --- 0.4 1.6 3.33 Diagnosis

non-regenerative

anemia

good regenerative

anemia

highly regenerative

anemia

SPECIES VARIATIONS: IN CLINICALLY HEALTHY ANIMALS

- Dogs. Low number of reticulocytes (<1%), aggregate only

- Cats Two types of reticulocytes:

5-36

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.

SPECIES VARIATION: in anaemicanimals

- Canine. Strong reticulocyte response in regenerative anaemias.

Aggregated reticulocytes (indicate recent response)

5-37

Aggregated reticulocytes (indicate recent response)- Feline.

Punctated reticulocytes (indicate response to anaemia occurring 3-4 weeks previously)

- Ruminants and horses. Reticulocytes may not appeareven in very severe anaemias