A. Manual diluting method.
Using a diluting fluid that destruct all blood cells
leaving only RBCs intact.
With the aid of a hemocytometer, counts the
number of cells expressed in millions per micro
liter of blood.
Add 2 ml of hayem’s solution to clean
and dry tube.
Add 10 µl of the sample (whole anti-
coagulated blood) and mix.
Dilution factor: 1:200
Charge the hemocytometer between the
cover slip and the counting chamber.
Leave the cells to settle down.
The depth factor is 110
Count the RBCs in 5 medium squares (4 at the
4 corners & 1 in the center) inside the central
Count those cells in L-shape manner
Erythrocytes / µl of blood= cells counted per
square х 10 (depth) х 5 х200(dilution)
Erythrocytes / µl of blood= cells counted per
square х 10 (depth)х 25х200 (dilution)
= …………………. Cells / µl or cells / mm3
B. Other automatic methodsB. Other automatic methods::
Electronic cell counter
Flow cell cytometers : depends on laser
RBCS MORPHOLOGYRBCS MORPHOLOGY
AND ABNORMALITIESAND ABNORMALITIES
RBCs morphologyRBCs morphology::
Flexibility, due to actin and
Mamalian RBCs are non
Poultry RBCs are elliptical
Fish RBCs are round
Poultry RBCs are elliptical nucleated.
Fish RBCs are round nucleated.
Dog Cat Equine Bovine Sheep Goat
Size 7µm 5.8 µm 5.7 µm 5.5 µm 4.5 µm < 4 µm
Central palor V. clear V.
Marked Common Rare in health and disease
Mild Common Characteristics
Pokilocytosis Slight Characteristics
Abnormalities in RBCs morphology:Abnormalities in RBCs morphology:
Abnormalities in size (Anisocytosis):
variation in size of cells due to presence of micro &/or
Macrocytes among normal cells.
Macrocytosis (↑MCV) : 1- True macrocytosis (vit. B12 and
folate deficiency) erythrocyte maturing factors
2-Pseudo macrocytosis (regenerative response of bone
marrow in case of blood loss.)
Microcytosis (↓MCV): iron deficiency or failure in
absorption or utilization of iron ( deficiency of Cu, Vit
C, vit. B6
Abnormalities in aggregation:
Agglutination: RBCs aggregated in the form of Clumps or clusters
( not in chains)
Causes :- Autoimmune mediated diseases).
Staphylococcal infection ( Protein A)
Rouleaux: RBCs arranged in the form of chain, like stock of
Normal in horse and to a Mild degree in dog and increase in Tumors,
infection and inflammation (hyperfibrinogenemia)
Rare in ruminant either in health or disease conditions
How can they differentiated
Abnormalities in RBCs shape:Abnormalities in RBCs shape:
Acanthocyte ( spur cellAcanthocyte ( spur cell))
(Irregular spine, uneven in size, numbers, diameter and
Causes:- Irregular projections , formed when
erythrocyte membrane contain excess cholesterol
compared to phospholipids,
Incidence:- common in severe liver disease.
Creanation – Echinocyte – burr cell:
(blunt to sharp projections, regularly distributed, even in
size and diameter)
Causes and incidence: in case of renal
insufficiency, uremia, artifact in lab.
Techniques(due to slow air drying)
Target cell ( codocytes) or leptocytes (folded
( centeral hemoglobinized area separated from pereferal
hemoglobinized area by clear zone)
due to increased surface Area : volume ratio membrane
folding and target cell formation
Causes:- Occurs from low Hb content as in case of iron
deficiency anemia, liver disease, regenerative
Seen in dog with increasersd serum cholesterol
Tear drop cell (Dacrocytes):
pear shaped with elongated blunt or pointed projection.
Causes:- Is thought to be due to distortion of RBCs as they
travel through vasculature of abnormal B.M or spleen.
Incidence:- myelofibrosis, metastatic tumor in the bone
marrow, extramedullary hematopoiesis, dyserythropoiesis,
Tear drop cell (Dacrocytes):Tear drop cell (Dacrocytes):
Sickle cell ( Drepanocyte):
Elongated, Fusiform, Spindle shaped or Crescent , and lack
Due to polymerlization of Hb
Causes:- Occurs in
of hemoglobin gene).
Spherocytes: Small darkly stained RBC
that loss central pallor
Incidence:- 1- Characteristic in IMHA
(Due to incomplete phagocytosis)
2- Hereditary spherocytosis.
Schistocyte or Keratocyte:
are red cell fragments which are formed when
fibrin strands come in contact with
circulating red cells. The strands cut a small
piece from the original cell.
Incidence:-Several microangiopathic diseases,
including disseminated intravascular
coagulation, intravascular truma and vascular
Stomatocytes : - Red cells in which the central pallor
is straight or appears as a curved rod-shaped slit.
The cells appear as "smiling face" or fish mouth.
Incidence:- occurs in hereditary stomatocytosis, liver
disease and may be as artifact in thick blood film.
Abnormalities in Hb contentAbnormalities in Hb content::
( structures in or on RBCs):
Howell- jolly bodies.
Parasites, babesia and anaplasma.
RBCs inclusionsRBCs inclusions::
Invivo aggregation of ribosoms
into small basophilic
Incidence:- occurs in lead
poisoning in Dog and
responsive anemia in
Howell- jolly bodies:- nuclear reminants in
erythrocytes ( small, round, dark blue inclusions of
Heinz bodies:Heinz bodies: Appear asAppear as small, eccentric, pale refractile
structure within RBC membrane and commonly seem to protrude
slightly from RBC marigin. ( with routine stain(
It called refractile body in cat and reach 10 %
Causes:- oxidative denaturation of Hb.by
oxidative drugs and compound ( Onion, garlic and
Zn & Cu toxicity.
While by vital stains ( NMB, brillent cresyl blue)
Appear as blue structures.
Babesia in RBCsBabesia in RBCs
Babesia in RBCs using Wright’s stain.
RBC indices include:
Mean Corpuscular Volume (MCV(
Mean Corpuscular Hemoglobin (MCH(
Mean Corpuscular Hemoglobin Concentration
RBC Distribution Width (RDW(
Mean cell volume
MCV is average size of RBC
MCV = Hct x 10
If 60-77 fL, normal range(canine), RBCs
If < 60 fL are microcytic
If > 77 fL are macrocytic
Not reliable when have marked
MCH is average weight of hemoglobin
MCH = Hgb x 10
Unite is pg
Normal canine range is 19-24 pg
MCHC is average hemoglobin
concentration per RBC
MCHC = Hgb x 100
If MCHC is normal, cell described as
If MCHC is less than normal, cell
described as hypochromic
There are no hyperchromic RBCs
Normal canine range is 32-36
Most automated instruments now
provide an RBC Distribution Width
An index of RBC size variation
May be used to quantitate the amount of
anisocytosis on peripheral blood smear
Increase in case of regenerative anemia
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