Red cell countRed cell count

MethodsMethodsA. Manual diluting method.

Principle: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.

ReagentsReagents

Gower’s solution: Sod. Sulphate, Glacial acetic acid and D.W

Hayem’s solution: mercuric chloride, NaCl, sod. Sulphate and

D.W. Physiological saline (0.85%)

EquipmentsEquipments::

HemocytometerCover slipPipettes

ProceduresProcedures::1. Dilution:

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

2. Charging

Charge the hemocytometer between the cover slip and the counting chamber.

Leave the cells to settle down.The depth factor is 1\10

3. Counting:

Count the RBCs in 5 medium squares (4 at the 4 corners & 1 in the center) inside the central large square.

Count those cells in L-shape manner

16 Tertiary squares

Reading (counting)Reading (counting)????? Cell /square.

CalculationCalculation::Erythrocytes / µl of blood= cells counted per

square х 10 (depth) х 5 х200(dilution)

ORErythrocytes / µl of blood= cells counted per

square х 10 (depth)х 25х200 (dilution)

= …………………. Cells / µl or cells / mm3

B. Other automatic B. Other automatic methodsmethods : :

Electronic cell counterFlow cell cytometers : depends

on laser lights.

RBCS MORPHOLOGY RBCS MORPHOLOGY AND ABNORMALITIESAND ABNORMALITIES

RBCs morphologyRBCs morphology::

Biconcave discFlexibility, due to actin and spectrinMamalian RBCs are non nucleated.

Poultry RBCs are elliptical nucleated.

Fish RBCs are round nucleated.

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. Slight

Lack SLight

Rouleux mild degree

Marked

Common

Rare in health and disease condition

Anisocytosis

Uniform size

Mild Common Characteristics

Pokilocytosis

Slight Characteristics

Abnormalities in RBCs Abnormalities in RBCs morphology: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

Variation of sizeVariation of size::

Polychromatophilic red blood cell , macrocytePseudomacrocytosis (reticulocyte)

True macrocytosisCauses???Pernicious anemia

AnisocytosAnisocytosis is

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

coins.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 Abnormalities in RBCs shape:shape:

(Pokilocytosis)(Pokilocytosis)Acanthocyte ( spur cellAcanthocyte ( spur cell))

(Irregular spine, uneven in size, numbers, diameter and distribution)

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)

Acanthocyte

Ecchinocyte

Target cell ( codocytes) or leptocytes (folded cell):

( 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

Anemia.

Seen in dog with increasersd serum cholesterol Level.

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, megaloblastic anemias, 

Tear drop cell (Dacrocytes):Tear drop cell (Dacrocytes):

Sickle cell ( Drepanocyte):Elongated, Fusiform, Spindle shaped or Crescent , and lack

central palor.

Due to polymerlization of Hb

Causes:- Occurs in

Hemoglobinopathies (mutation

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 neoplasm (hemangiosarcoma)

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 Abnormalities in Hb contentcontent::HypochromaciaPolychromacia.

Hypochromacia

RBCs inclusions ( structures in or on RBCs):

Basophilic stippling.Heinz bodies.Howell- jolly bodies.Parasites, babesia and anaplasma.

Nucleated RBCs.

RBCs inclusionsRBCs inclusions::Basophilic stippling.Invivo aggregation of ribosoms

into small basophilic granules

Incidence:- occurs in lead poisoning in Dog and responsive anemia in ruminant .

Howell- jolly bodies:- nuclear reminants in erythrocytes ( small, round, dark blue inclusions of variable Size

Shape

Site

Incidence:- spleenic dysfunction Spleenectomy regenerative anemia

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 % normally .

Causes:- oxidative denaturation of Hb.by oxidative drugs and compound ( Onion, garlic and Zn & Cu toxicity. it increase

susceptibility for

Hemolysis.

While by vital stains ( NMB, brillent cresyl blue)

Appear as blue structures.

Babesia in RBCsBabesia in RBCs

Babesia in RBCs using Wright’s stain.

Nucleated RBCs ( Erythroblastosis)Nucleated RBCs ( Erythroblastosis)

associated with regenerative anemia, spleenic dysfunction

RBC indices include: Mean Corpuscular Volume (MCV)

Mean Corpuscular Hemoglobin (MCH)

Mean Corpuscular Hemoglobin Concentration (MCHC)

RBC Distribution Width (RDW)

MCVMCVMean cell volumeMCV is average size of RBCMCV = Hct x 10

RBC (millions) If 60-77 fL, normal range(canine),

RBCs considered normocyticIf < 60 fL are microcyticIf > 77 fL are macrocytic Not reliable when have marked

anisocytosis

MCHMCHMCH is average weight of

hemoglobin per RBC. MCH = Hgb x 10 RBC (millions) Unite is pgNormal canine range is 19-24 pg

MCHCMCHCMCHC is average hemoglobin

concentration per RBC MCHC = Hgb x 100

Hct (%) If MCHC is normal, cell described

as normochromic If MCHC is less than normal, cell

described as hypochromic There are no hyperchromic

RBCsNormal canine range is 32-36

RDWRDWMost automated instruments now

provide an RBC Distribution Width (RDW)

An index of RBC size variationMay be used to quantitate the

amount of anisocytosis on peripheral blood smear

Increase in case of regenerative anemia