White Blood Cell Differential Count Last update 17-12-2014

Leukocytes (WBCs) 5 types of WBCs characterized as granular or agranular all function in defense average 4000 -11000 WBCs/mm3 of blood variable “life” span depending on type of WBC- days (neutrophils) to decades (lymphocytes).

Differential Count & Functions of WBCs Neutrophils - 50-70%, function in acute bacterial infections; phagocytic. Eosinophils – 2-4%: active against parasites & elevated in allergic reactions; destroy antibody-coated antigens by phagocytosis. Basophils – 0-1% : release chemicals mediators (histamine, heparin) during tissue inflammation.

Differential Count & Functions of WBCs Lymphocytes – 25-40%: function in “immunity” – specific resistance to disease Monocytes – 3-8%: function in chronic bacterial infections; migrate into tissues to become “macrophages”

MYELOBLAST PROMYELOCYTE MYELOCYTE No Granules, N/C ratio 6/1, Pale blue cytoplasm , ~ 2% of normal marrow, Not present in blood of Healthy Persons. Usually larger than MYELOBLAST, Granules are large & azurophilic, N/C ratio is 4/1. : Round or Oval cell with a round nucleus, Granules are either primary azurophilic or secondary specific according to WBC type: (Neutrophil, Eosinophil or Basophil). MYELOBLAST: 16 um, 2 to 6 Nucleoli, No Granules, N/C ratio 6/1, Pale blue cytoplasm , ~ 2% of normal marrow, Not present in blood of Healthy Persons. PROMYELOCYTE: Usually larger than MYELOBLAST, Granules are large & azurophilic, N/C ratio is 4/1. MYELOCYTE: Round or Oval cell with a round nucleus, Granules are either primary azurophilic or secondary specific according to WBC type: (Neutrophil, Eosinophil or Basophil). META-MYELOCYTE: Kidney-shaped nucleus STAB or BAND FORM: Smaller cells with a U-shaped nucleus

STAB or BAND FORM Kidney-shaped nucleus META-MYELOCYTE STAB or BAND FORM Kidney-shaped nucleus Smaller cells with a U-shaped nucleus MYELOBLAST: 16 um, 2 to 6 Nucleoli, No Granules, N/C ratio 6/1, Pale blue cytoplasm , ~ 2% of normal marrow, Not present in blood of Healthy Persons. PROMYELOCYTE: Usually larger than MYELOBLAST, Granules are large & azurophilic, N/C ratio is 4/1. MYELOCYTE: Round or Oval cell with a round nucleus, Granules are either primary azurophilic or secondary specific according to WBC type: (Neutrophil, Eosinophil or Basophil). META-MYELOCYTE: Kidney-shaped nucleus STAB or BAND FORM: Smaller cells with a U-shaped nucleus

SEGMENTED GRANULOCYTES Neutrophils have pink cytoplasm with fine evenly-distributed neutrophilic granules 2 to 6 nuclear segments

Eosinophils have bi-lobed nuclei & orange Course granules SEGMENTED GRANULOCYTES

Basophils have bi-lobed nuclei & deep blue granules SEGMENTED GRANULOCYTES

Agranulocytes Monocytes Lymphocytes Pleuripotential stem cell Multipotential stem cell Monocytes Lymphocytes

Monoblasts Monocytes Bigger than myeloblasts Largest blood cells Usually kidney-shaped nuclei

Lymphoblast Lymphocytes Nucleus fills the cell Lymphocytes Small LC have a round, nucleus that fills the cell & have size of RBC Large LC is double the size

White Blood Cell Differential Count Usually performed by visual examination of blood film that are prepared on slides by the spread technique

Equipments: Smooth glass spreader Glass slides Capillary tubes Light microscope Mechanical or electronic counter Leishman stain

Specimen Fresh blood with no anticoagulant EDTA –anticoagulated blood

Procedure: Place small drop of blood on the center of clean dry glass slide about 1 cm from the end Then without delay, a spreader in front of the drop at angle of 30o to the slide and move it back to make contact with the drop With a steady movement of the hand, spread the drop of the blood along the slide

Procedure… 4-The film should be allowed to be dry in the air 5-The film should be labeled immediately after it had been dried by pencil 6-Using Leishman’s stain, flood the slide with the stain for 2 min 7-Add double the volume of water and stain the film for 5-7 min 8-Wash it in a stream of water.

Procedure… 9-After the back of the slide has been wiped clean, sit it upright to dry 10-Examine the film using x40 objective in a strip, running the whole length of the film 11-Avoid the lateral edges 12-Inspect the film from head to tail 13-Count 200 cell or more to get an accurate result using counter.

Absolute count Absolute Leukocytes count: = Relative count * Total Count e.g. if Neutrophils = 55% , Total Count = 6000 C/µl So, Absolute neutrophil count= 0.55 * 6000 = 3300

White blood cell count 4.0–11.0 × 109/L Differential white cell count Neutrophils: 2.0–7.0 × 109/L (50–70%) Lymphocytes: 1.0–3.0 × 109/L (25–40%) Monocytes: 0.2–1.0 × 109/L (3–8%) Eosinophils: 0.02–0.5 × 109/L (2–4%) Basophils: 0.02–0.1 × 109/L (0-1%)

Correcting WBC for nucleated RBC Corrected WBC= total cell counted – (total cell counted * no. of nRBC\100)