Red Blood Cell (RBC)and

White Blood Cell (WBC)Counts

Blood

Important Blood Terminology• Whole blood: Composed of plasma (fluid portion of blood) and

formed elements (blood cells and platelets).• Formed Elements: Red blood cells, white blood cells, and

platelets. • Plasma: Fluid portion of blood that contains proteins, ions,

nutrients, hormones, antibodies, metabolites, enzymes, etc.• Serum: Similar to plasma, but without clotting factors such as fibrin. • Hematocrit: Percentage of total blood volume that is occupied by

red blood cells; also called packed red cell volume.• Red cell count: The number of red blood cells per cubic milliliter

(mm3) of whole blood. 1 mm3 = 1 µL.• Total white cell count: The total number of all white blood cells per

cubic milliliter (mm3) of whole blood. This number does not distinguish between different white cell types.

• Differential white cell count: The relative numbers of the five types of white blood cells. The numbers are reported as percentages.

Taken from Diggs, L.W., Sturm, D., and Bell, A. The Morphology of Human Blood Cells, 5th Edition, Abbot Laboratories, Abbot Park, Illinois.

Composition of Blood

• Plasma: Fluid portion of blood (~55%)– Straw-colored– Contains water, ions, nutrients, hormones,

antibodies, enzymes, metabolites, and proteins.

– Na+ and Cl– are very high in concentration and contribute most to plasma osmolality.

• Formed Elements: Cellular component of blood (~45%)– Red blood cells (erythrocytes)– White blood cells (leukocytes)– Platelets (thrombocytes)

Plasma Proteins (7–9% of plasma)

• Albumins (60–80% of all plasma proteins)– Smallest in size– Produced by liver– Also important in contributing to plasma

osmotic pressure– Many hormones and ions are bound to

albumin

Plasma Proteins (7–9% of plasma)• Globulins

– Alpha globulins– Beta globulins

• Both alpha and beta globulins are produced by the liver and function to transport lipids and fat-soluble vitamins.

– Gamma globulins• Antibodies produced by lymphocytes

• Fibrinogens– Produced by the liver– Function in blood clotting

Red Blood Cells• Synonym: Erythrocytes• Most numerous cells in blood

– Males: 4.5–6.0 million/mm3

– Females: 4.0–5.5 million/mm3

• Flattened biconcave discs• Lack nucleus and mitochondria• Each RBC has about 280 million

hemoglobin molecules• Lifespan: 120 days

White Blood Cells• Synonym: Leukocytes• 4,500 – 11,000 / mm3

• Contain nucleus and mitochondria• Capable of amoeboid movement

– Diapedesis: the movement of leukocytes through capillary walls.

• Classified based on stained appearance:– Granular leukocytes– Agranular leukocytes– Lymphocytes

Leukocytes• Granular leukocytes

– Eosinophils (1–3%)– Basophils (<1%)– Neutrophils = Polymorphonuclear neutrophils

(PMN) (50–60%)– Lifespan 12 hours to 3 days

Leukocytes Continued• Agranular leukocytes

– Monocytes (3–9%)• Largest of leukocytes• Kidney-shaped nuclei• Lifespan of 100–300 days

– Lymphocytes (25–33%)• Small cells• Round nuclei• Small cytoplasm• T Lymphocytes• B Lymphocytes

– Plasma cells (produce antibodies)

Platelets• Synonym: Thrombocytes• 130,000–360,000 /mm3

• Smallest of formed elements• Cytoplasmic fragments of megakaryocytes

in bone marrow• Lack nuclei• Capable of amoeboid movement• Important in the formation of bulk of the

clot during blood clotting• Lifespan of 5–9 days

Basic Procedure for RBC and WBC

• Obtain whole blood in a tube that contains EDTA (Na+ or K+ salt).

• EDTA– Ethylenediaminetetraacetic acid

• EDTA chelates Ca2+ and thus prevents the formation of blood clots.

• This blood sample may be used as the starting point for both the red blood cell (RBC) count and white blood cell (WBC) count procedures.

Red Blood Cell Count• Mix blood in tube well.• Dilute whole blood in Hayem’s solution.

– 1:200 dilution• Hayem’s Solution

– Isotonic with blood– Contains

• 0.5% NaCl• 2.5% Na2SO4 (to prevent clump formation)• 0.25% HgCl2 (mercuric chloride) (preservative)

• Apply a small volume (~10 µl) of diluted blood to one side of a hemacytometer.

Hemacytometer

Hemacytometer

Hemacytometer

Hemacytometer

Boundary Rule

Calculation of RBC

AFactorDilution Factor Depth Count

mmN

3

××=

• N/mm3: Number of cells per cubic mm• Count: Total number of cells counted• Depth Factor: 10• Dilution Factor: 200• A: Area used for count in mm2 = 0.2 mm2

• Thus, for RBC: Multiply your total count by 10,000.

Reference Ranges for Human RBC Counts

• Adult Male: 4.5–6.0 x 106 /mm3

• Adult Female: 4.0–5.5 x 106 /mm3

• Newborn: 5.0–6.3 x 106 /mm3

White Blood Cell Count• Mix blood in tube well.• Dilute whole blood in Turk’s solution.

– 1:100 dilution• Turk’s Solution

– Red blood cells burst in this solution, but white blood cells remain intact and are stained.

– Contains• 1% Acetic acid• 0.01% Gentian violet (crystal violet) (stain)

• Apply a small volume (~10 µl) of diluted blood to one side of a hemacytometer.

Calculation of WBC

AFactorDilution Factor Depth Count

mmN

3

××=

• N/mm3: Number of cells per cubic mm• Count: Total number of cells counted• Depth Factor: 10• Dilution Factor: 100• A: Area counted in mm2 = 4 mm2

• Thus, for WBC: Multiply your total count by 250.

Reference Ranges for Human WBC Counts

• Newborn: 9,000–30,000 /mm3

– Average: 18,000 /mm3

• 1 Year: 6,000–14,000 /mm3

– Average: 11,000 /mm3

• 6 Years: 4,500–12,000 /mm3

– Average: 8,000 /mm3

• Adult: 4,500–11,000 /mm3

– Average: 7,400 /mm3