IMMUNOLOGY PROJECT

TOTAL LEUKOCYTE COUNT AND DIFFERENTIAL LEUKOCYTE COUNT

SUBMITTED BY: AYUSHI UE111012 DHAARNA UE111013 DIVESH UE111014 GARIMA UE111015 GAURAV UE111016 GAURAV UE111017 SUBMITTED TO: MS. ANUPRIYA MINHAS GAURAV UE111018 GURVIKRAM UE111019 HARSHEEN UE111020

AIM: To perform a) Total leukocyte count b) Differential leukocyte count of the given blood sample. REAGENTS AND EQUIPMENTS: Sample microscope Hemocytometer chamber Cover slip Blood sample Turks fluid composition: 0.025 gm Gentian violet 2 ml Glacial Acetic Acid 100 ml Distilled water. THEORY: HAMOCYTOMETERThe hemocytometer counting chamber is used for cell counting. It is constructed so that the distance between the bottom of the cover slip and the surface of the counting area of the chamber is 0.1 mm. The surface of the chamber contains two square ruled areas separated by an H-shaped moat. These two squares are identical, allowing the technologist to duplicate the cell count. Each has a total area of 9 mm2 (1 mm on each side). These squares are divided into nine primary squares with an area of 1mm2. The four corner primary squares are used when counting leukocytes. (If the WBC is very low, you would count all 9 squares.) These 4 large corner squares contain 16 smaller secondary squares, each with an area of 0.04 mm2. All 25 secondary squares of the center primary square are used to count platelets, and each of these 25 squares is further divided into 16 smaller tertiary squares (see figure). Cells which lie on LEFT and TOP boundary lines of the squares are counted. Cells which lie on the RIGHT and BOTTOM boundary lines of the squares are NOT counted. (This is to make cell counts statistically accurate.) PRINCIPLE:

Part B

MATERIALS REQUIRED:
The composition of Giemsa stain: Giemsa powder 0.3 gm Glycerin 25 ml Methyl alcohol 25 ml this makes stock solution and before use it has to be diluted by adding 1 ml (stain) to 9 ml of buffered water THEORY A total white blood cell count is not necessarily indicative of the severity of a disease,

since some serious ailments may show a low white cell count. For this reason, a differential

white cell count is performed. A differential white cell count consists of an examination of blood to determine the presence and the number of different types of white blood cells. This study often provides helpful information in determining the severity and extent of an infection, more than any other single procedure used in the examination of the blood. The role of white blood cells, or leukocytes, is to control various disease conditions. Although these cells do most of their work outside the circulatory system, they use the blood for transportation to sites of infection. Leukocyte are classified into 2 types namely

1. Granulocyte-with granules 2. Agranulocyte- without granules The granulocyte are-Neutrophills Eosinophills Basophills The agranulocyte are-Monocytes Lymphocytes NEUTROPHILS. - 1.Neutrophils are produced by hematopoiesis in the bone marrow. 2. The neutrophil has a multilobed nucleus and a granulated cytoplasm that stains with both acid and basic dyes; 3. Neutrophils, constitute 50%70% of the circulating white blood cells

EOSINOPHILS: 1.Eosinophils, like neutrophils, are motile phagocytic cells that can migrate from the blood into the tissue spaces. 2. They play a role in the defense against parasitic organisms . 3.The secreted contents of eosinophilic granules may damage the parasite membrane.

4.The eosinophil has a bilobed nucleus and a granulated cytoplasm that stains with the acid dye eosin red BASOPHILS: 1.Basophils are nonphagocytic granulocytes that function by releasing pharmacologically active substances from their cytoplasmic granules. 2.These substances play a major role in certain allergic responses. 3.The basophil has a lobed nucleus and heavily granulated cytoplasm that stains with the basic dye methylene blue. LYMPHOCYTE.-1.The function of lymphocytes is also unknown, but it is believed that they produce antibodies and destroy the toxic products of protein metabolism. 2.The cytoplasm of a lymphocyte is clear sky blue, scanty, with few unevenly distributed. 3. The nucleus is generally round, oval, or slightly indented.

MONOCYTE.-1.The monocyte, the largest of the normal white blood cells, destroys bacteria, foreign particles, and protozoa. 2.Its color resembles that of a lymphocyte, but its cytoplasm is a muddy grayblue . 3.The nucleus is lobulated, deeply indented or horseshoe-shaped, and has a relatively fine chromatin structure.
PRINCIPLE: PROCEDURE: 1. a) b) c) d) e) f)
Preparation of the slide:

Place a drop of blood in the centre of a clean glass slide 1 to 2 cm from one end. Place another slide with smooth edge at an angle of 30-45 0 near the drop of blood. Move the spreader backward so that it makes contact with drop of blood. Then move the spreader forward rapidly over the slide. A thin peripheral blood film is thus prepared. Dry it and stain.

Fig. B1 -Staining procedure 2. Staining procedure: a) The blood film is fixed with methyl alcohol for 2 minutes. b) Pour Giemsa stain diluted 1:9 with buffer over the smear for 8-10 minutes. c) Wash off with buffer and dry. The Count : The dry and stained film examined without a coverslipe under oil immersion objective .for differential leukocyte counts choose an area where the morphology of the cells is clearly visible .do differential count by moving the slide in area including the central and peripheral and the smear .a total of 200 cells should be counted in which every white cell seen must be recorded in a table under the following heading: Neutrophile, Eosinophile, Basophile, Lymphocyte, and Monocyte then find the percentage of each type.