Undergraduate Course inVeterinary Clinical Pathology

Socrates-Erasmus Programme

Haematology
LECTURE 4.
4 TESTS IN HAEMATOLOGY: THE
HAEMOGRAM

4-1

OVERVIEW
1. Packed cell volume (PCV)/haematocrit (Hct) and
total proteins
2. Erythrocyte (RBC) count, haemoglobin (Hb) and
indices
3. Leukocyte total and differential count
4. Platelet count, morphology and Mean Platelet
Volume (MPV)
5. Histograms
6. Blood smear evaluation

4-2

1.PCV/Hct AND TOTAL PROTEINS

4-3

PACKED CELL VOLUME (PCV)

PCV is the ratio of erythrocyte volume to whole blood volume
- PCV is measured by whole blood centrifugation
(microhaematocrit method)
-Haematocrit is calculated from RBC count and mean cell
volume by electronic cell counters
In clinical terms PCV = Haematocrit
They are used to detect polycythaemia and anaemia
4-4

ADVANTAGES OF PCV MEASUREMENT

-It is a very simple, precise and accurate method
-It provides more than just a PCV (see next slide)
Erythrocytic area

Plasma area

Buffy coat
4-5

PRACTICAL USES OF DIFFERENT SECTIONS

OF CAPILLARY TUBE

ERYTHROCYTIC SECTION

BUFFY COAT

PLASMA SECTION

Packed Cell Volume

determination

-Increase suggests
gg
leukocytosis
y
or thrombocytosis
y
-May be used to make concentrated WBCs smears
and to aid detection of Microfilaria
-Analysis of total plasma proteins/ fibrinogen
-Its colour can provide clinical information:
icterus, carotene (cows)
haemolysis
lipaemia
4-6

CAPILLARY TUBE SECTIONS AND PLASMA

COLOURS icterus or
carotene
dehydration (cattle) haemolysis

Leukocytosis or
lipaemia thrombocytosis

plasma
buffy
coat

erythrocytes

4-7

TOTAL PLASMA PROTEINS (TPP)

Can be determined from the plasma at the top of a centrifuged
capillary tube using a refractometer
high concentration of glucose/urea

Sources
of error

lipaemia or haemolysis
values <25 g/L or >95 g/L

4-8

REFERENCE VALUES FOR PCV AND TPP IN

DIFFERENT SPECIES

Dog
PCV(L/L)
TPP (g/L)
( /L)

Cat

0.37-0.55 0.24-0.45
54-77

54-78

Horse
0.35-0.52
52-79

Cow
0.26-0.42
67-75

These values are only indicative. Each laboratory should

have its own reference ranges.

4-9

INTERPRETATION OF RESULTS

1. Increase in PCV.............. POLYCYTHAEMIA

+ TPP
dehydration

2. Decrease in PCV.............. ANAEMIA

+ TPP
blood loss

4-10

2. ERYTHROCYTE (RBC)
COUNT HAEMOGLOBIN (Hb)
COUNT,
AND INDICES

4-11

RBC COUNT AND Hb CONCENTRATION

-Can be determined:
- by manual methods
- by haematological analysers:
impedance counters
laser
l
cellll counters
t
quantitative buffy coat analysers
-Give similar clinical information as the PCV
- Are used to calculate RBC indices
4-12

RBC INDICES
MCV (MEAN CELL VOLUME)
MCV (fl) = PCV(L/L) x 1000 / RBC COUNT (1012/L)
Indicates the average size of RBCs:

indicates MACROCYTOSIS

values within reference range indicates NORMOCYTOSIS

indicates MICROCYTOSIS
4-13

MCHC (MEAN CELL HAEMOGLOBIN

CONCENTRATION)
MCH (MEAN CELL HAEMOGLOBIN)
MCHC (g/L) = Hb(g/L) / PCV(L/L)
MCH (pg) = Hb(g/L) / RBC(1012/L)
Indicates the average concentration of Hb in RBCs:
indicates HYPOCHROMASIA
values within reference range indicates NORMOCHROMASIA

4-14

RED CELL DISTRIBUTION WIDTH (RDW)

A numeric representation of the variability in RBC size (RBC
anisocytosis).
usually indicates

Increased RDW
and
d MCV

similar RBC size

Regenerative anaemia

different RBC size, increase RDW

4-15

3. LEUKOCYTE TOTAL AND

DIFFERENTIAL COUNT

4-16

WBC TOTAL COUNT

Can be determined:
manually by haemocytometer and microscope
automated by haematological analysers
byy estimation from a blood smear
-100x magnification
Mean of 10 fields x 100 = WBC x 109/L
-400x magnification
Mean of 10 fields x 1,500 = WBC x 109/L
4-17

-LEUKOCYTE ESTIMATION IN A BLOOD

SMEAR (400X):

Normal number

Increased

Decreased
4-18

DIFFERENTIAL LEUKOCYTE COUNT

Can be determined:
from a stained blood smear
automated
automated by some haematological analysers
analysers,
but these need prior validation

4-19

HOW TO PERFORM A LEUKOCYTE

DIFFERENTIAL COUNT IN A STAINED BLOOD
SMEAR: BATTLESHIP METHOD

4-20

TYPES OF LEUKOCYTES

- GRANULOCYTES:
Neutrophils, Eosinophils, Basophils
- MONOCYTES
- LYMPHOCYTES

4-21

Normal neutrophils and a lymphocyte, dog

4-22

Small lymphocytes, dog

Small and medium lymphocyte, cow

4-23

Normal neutrophil and monocyte, dog

4-24

Normal eosinophil and basophil, cat

4-25

Normal WBCs in different species, some examples

(1)

(2)

(4)

(3)

4-26

REPORTING A DIFFERENTIAL COUNT

By relative % of each WBC type

(INCORRECT)
Ways to
report the
values
By absolute number of each WBC type
(CORRECT)
(%of each cell type x total WBC number)
4-27

EXAMPLE
Dog n1 : 70% segmented and 10.0 x 109/L WBCs
Dog n2: 70% segmented and 1.0 x 109/L WBCs
Dog n3 : 70% segmented and 50.0 x 109/L WBCs
%
Segmented neutrophils

Absolute segmented
neutrophils x 109/L

Dog n1 : 70 (NORMAL)

7.0 NORMAL

Dog n2: 70 (NORMAL)

0.7 (DECREASED)

Dog n3 : 70 (NORMAL)

35.0 (INCREASED)
4-28

INTERPRETING DIFFERENTIAL RESULTS

in a particular leukocyte

Neutrophilia
Eosinophilia
Basophilia
Lymphocytosis
Monocytosis

in a particular leukocyte

Suffix penia
(i.e. Lymphopenia)
4-29

4. PLATELET COUNT,
MORPHOLOGY AND MPV
EVALUATION

4-30

PLATELET COUNT (PLT)

Three methods can be used:
- A manual count
- An estimated count from a blood smear (average =15-20
platelets in 10 microscope fields at 1,000x)
1 000x)

- An automated cell count

PLT counts should be performed as soon as possible
after collection of blood, to avoid clumping
4-31

PLATELET MORPHOLOGY AND MPV

(MEAN PLATELET VOLUME) EVALUATION
Can detect:
- platelet clumping at the feathered edge of blood smear
(pseudo-thrombocytopenia)
- platelets with large cell volume (high MPV)
- platelets with small cell volume (low MPV)

4-32

PLATELET
AGGREGATE

PLATELETS WITH
DIFFERENT SIZE

4-33

5 HISTOGRAMS
5.

4-34

Histograms are provided by automated analyzers and represent

the distribution of the sizes of a determined cell population.
dog

cat

horse

Examples of
histograms in
different species
obtained from an
impedance cell
counter

4-35

6. BLOOD SMEAR EVALUATION

4-36

HOW TO MAKE A BLOOD SMEAR

4-37

BLOOD SMEAR STAINING

Romanowsky stains are usually used for routine staining.
This group of stains includes:
- May Grunwald Giemsa
- Diff-quik
- Wright
- Leishman

4-38

A BLOOD SMEAR SHOULD PROVIDE:

An area where cell morphology can be observed with optimal
detail because cells:
-are
are in a monolayer (about 50% are in contact with each
other)
-do not overlap each other (base/head/beginning of the
smear)
-are not damaged or distorted (feathered edge/end of smear)
4-39

MAIN PARTS OF A BLOOD SMEAR

Base or head

Monolayer

Feathered edge

4-40

STEPS TO EVALUATE A BLOOD SMEAR

-Selection of monolayer area

At 100x magnification -Detection of platelet agreggation,

RBCs rouleaux/agglutination
-WBC
WBC number estimation/differential

At 400x magnification count

At 1000x magnification -Detailed evaluation of

RBCs, WBCs and platelets

4-41